Broad spectrum conjugate vaccine to prevent klebsiella pneumoniae and pseudomonas aeruginosa infections

ABSTRACT

The present invention is drawn to conjugates and vaccine compositions comprising a  Pseudomonas  flagellin or an antigenic fragment or derivative thereof linked to one or more  Klebsiella  surface polysaccharide antigens, such as  Klebsiella pneumoniae  O5 polysaccharide from serovars O1, O2a, O2a,c, O3, O4, O5, O7, O8 and 012. The present invention also provides serovar reagent strains to produce the conjugates and vaccine compositions and methods of inducing an immune response with the conjugates and vaccine compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. No.61/052,256, filed Sep. 18, 2014. The content of the aforementionedapplication is relied upon and is incorporated by reference in itsentirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant NumberW81XWH-15-2-0028 awarded by United States Army Medical Research andMaterial Command (USAMRMC). The government has certain rights in theinvention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablesequence listing submitted concurrently herewith and identified asfollows: One 58,497 Byte ASCII (Text) file named “seq_listing_ST25.txt,”created on Sep. 18, 2015.

FIELD OF THE INVENTION

The present invention generally relates at least to the fields ofmedicine, immunology, molecular biology and infectious diseases. Inparticular, the invention relates to novel conjugate vaccines fortreating or preventing invasive blood infections, urinary tractinfections, respiratory infections (including cystic fibrosis), woundinfections, central nervous system infections and burn infections aswell as nosocomial and community acquired infections caused byKlebsiella and Pseudomonas bacteria and septic shock.

BACKGROUND OF THE INVENTION

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are GramNegative Bacteria (GNB) that are among the most prevalent and virulentpathogens associated with wound infections in combat personnel. They cancause serious clinical syndromes including abscess formation,cellulitis, disseminated infection, and bacteremia leading toprogressive amputation, permanent impairment and death by septic shock.The growing proportion of Klebsiella pneumoniae and PA that aremulti-drug resistant (MDR) complicates treatment Immunoprophylacticmeasures against PA and Klebsiella pneumoniae can be effectiveirrespective of the antibiotic resistance phenotypes.

Klebsiella pneumoniae can express two virulence-associatedpolysaccharides (PS): a secreted cell-associated capsular polysaccharide(CPS) that coats the bacterium and a lipopolysaccharide (LPS) that formsthe outer leaflet of the outer-membrane. The polysaccharide portion ofKlebsiella pneumoniae LPS is comprised of a genus-specific conservedcore and a serotype specific polymer of O polysaccharide (OPS; FIG. 1)for which there are ˜9 recognized serotypes (Vinogradov E, J Biol Chem.2002; 277(28):25070-25081; Vinogradov E, Carbohydr Res. 2001;335(4):291-296).

Importantly, the prevalence of OPS types among clinical isolates ishighly restricted. Hospital based surveys of invasive infections haverevealed that four OPS serotypes (O1, O2a, O3 and O5) account for 60-80%strains causing infections in the USA and worldwide. By comparison,there are at least 80 identified CPS serotypes of which greater than 25are associated with invasive infections in humans in the USA (PodschunR, Clin Microbiol Rev. 1998; 11(4):589-603; Hansen D S, J ClinMicrobiol. 1999; 37(1):56-62); Trautmann M, Vaccine. 2004;22(7):818-821; Cryz S J, Jr., J Clin Microbiol. 1986; 23(4):687-690).Furthermore, the incidence and prevalence of invasive infectionsattributed to various CPS serotypes varies dramatically worldwide; CPStypes that are prevalent in one region, can be absent entirely in others(Cryz S J, Jr., J Clin Microbiol. 1986; 23(4):687-690), includingpotential areas of military deployment.

Despite envelopment by CPS, evidence has accumulated that Klebsiellapneumoniae LPS is accessible to antibody. LPS expression is required forprotection against the alternative pathway of the complement system(Merino S, Infect Immun. 1992; 60(6):2529-2535). Long-chain OPS polymersextend beyond the capsule surface and activate the alternativecomplement pathway at their distal ends, too far from the cell-surfaceto be functional (Tomas J M, Infect Immun. 1991; 59(6):2006-2011;Williams P, J Gen Microbiol. 1983; 129(7):2181-2191; Tomas J M, MicrobPathog. 1988; 5(2):141-147). Selective pressure for OPS expression hasbeen documented when KP is grown in human serum and absent when serumcomplement is heat-inactivated or KP is grown in broth culture (CamprubiS, Microb Pathog. 1992; 13(2):145-155). LPS expression has also beenassociated with establishment of invasive infections in animal models(Lawlor M S, Mol Microbiol. 2005; 58(4):1054-1073; Hsieh P F, PLoS One.2012; 7(3):e33155). Short-chain LPS is also likely to be antibodyaccessible, as several capsule types have been documented as permeableto antibody (Meno Y, Infect Immun. 1990; 58(5):1421-1428; Williams P, JMed Microbiol. 1988; 26(1):29-35); and LPS can become further exposed bythin and incomplete encapsulation.

Klebsiella pneumoniae CPS are important virulence factors thatantagonize non-specific opsonophagocytic uptake and capsule-deficientKlebsiella pneumoniae are highly attenuated (Williams P, J GenMicrobiol. 1983; 129(7):2181-2191; Domenico P, Infect Immun. 1994;62(10):4495-4499). However, expression of CPS inhibits bindinginteractions by Klebsiella pneumoniae adhesins with epithelial cells, animportant early step in infection; thus it is likely that CPS expressionis down-regulated in the early stages of infection (Favre-Bonte S,Infect Immun. 1999; 67(2):554-561; Hennequin C, Res Microbiol. 2007;158(4):339-347; Schembri M A, Infect Immun. 2005; 73(8):4626-4633).Numerous studies have supported the role of antibodies towardsKlebsiella pneumoniae LPS in protection against invasive KP infectionwith encapsulated strains. Antibodies to OPS antigen induced by activeimmunization with purified LPS (Tomas J M, Infect Immun. 1991;59(6):2006-2011; Clements A, Vaccine. 2008; 26(44):5649-5653; ChhibberS, Jpn J Infect Dis. 2004; 57(4):150-155), OPS:protein conjugates(Chhibber S, Indian J Exp Biol. 2005; 43(1):40-45; Chhibber S, Vaccine.1995; 13(2):179-184), killed whole-cells (Shimoguchi K., KansenshogakuZasshi. 1990; 64(12):1482-1492), acapsular mutants (Lawlor M S, InfectImmun. 2006; 74(9):5402-5407), or passive transfer with polyclonal(Clements A, Vaccine. 2008; 26(44):5649-5653) or monoclonal (Held T K,Infect Immun. 2000; 68(5):2402-2409) anti-LPS antibodies have protectedagainst fatal Klebsiella pneumoniae pneumonic and intraperitonealinfections in rodents. Parenteral immunization with formalin inactivatedwhole-cell encapsulated Klebsiella pneumoniae has protected againstinfection with the homologous encapsulated strain, and remarkablynegligible anti-CPS but robust anti-LPS antibody was detected, for whichthe level correlated well with protection (Shimoguchi K., KansenshogakuZasshi. 1990;64(12):1482-1492) Immunization with purified O1 LPS hasalso elicited protection against O1 strains expressing different capsuletypes (Tomas J M, Infect Immun. 1991; 59(6):2006-2011) Anti-Klebsiellapneumoniae O1 OPS monoclonal antibodies have demonstrated enhancedopsonophagocytosis of encapsulated strains (Held T K, Infect Immun.2000; 68(5):2402-2409), and protected against encapsulated Klebsiellapneumoniae when given by passive transfer (Rukavina T, Infect Immun.1997; 65(5):1754-1760). Partial protection has also been obtained byantibodies directed towards the core polysaccharide that is conservedamong Klebsiella pneumoniae, the diminished protection relative toanti-OPS is likely due however to steric hindrance for accessibility ofthe core polysaccharide to antibody in the context of long-chain OPS(Chen W H, Innate Immun. 2008; 14(5):269-278; Mandine E, Infect Immun.1990; 58(9):2828-2833).

Immune responses to Klebsiella pneumoniae outer membrane proteins (e.g.,iron regulated proteins, porins) have also protected (Chhibber S,Vaccine. 1995; 13(2):179-184; Serushago B A, J Gen Microbiol. 1989;135(8):2259-2268; Kurupati P, Clin Vaccine Immunol. Jan 2011;18(1):82-88). However, evidence suggests that LPS is the superiorvaccine target, as antibodies to purified OMP proteins did not protectas well as antibodies to non-encapsulated whole cell preparations thatincluded LPS (Serushago B A, J Gen Microbiol. 1989; 135(8):2259-2268)Immunization with Klebsiella pneumoniae capsular polysaccharides haveprotected against burn-wound Klebsiella pneumoniae infections in animalmodels (Cryz S J, Jr., J Infect Dis. 1984; 150(6):817-822), and passivetransfer with anti-capsule antibodies recapitulated the protection seenwith active vaccination (Cryz S J, Jr., Infect Immun. 1984;45(1):139-142). As anti-LPS antibodies are protective againstintraperitoneal (IP) and pneumonic Klebsiella pneumoniae infections inmice, they are also presumed to be protective against wound infectionscaused by Klebsiella pneumoniae.

Generating a CPS-based vaccine that would be effective againstpathogenic Klebsiella pneumoniae strains worldwide is not easilyaccomplished as the manufacture and establishment of acceptableimmunogenicity for all components of a ≧25 valent vaccine is a majorchallenge. A 24-valent Klebsiella pneumoniae CPS vaccine was shown to beimmunogenic in human trials (Edelman R, Vaccine. 1994;12(14):1288-1294). However, the levels varied dramatically amongserotypes, with some inducing only poor antibody levels. Importantly,antibody levels for most Klebsiella pneumoniae CPS types plunged withinthe 18 months of follow-up to pre-immune levels (Edelman R, Vaccine.1994; 12(14):1288-1294; Granstrom M, J Clin Microbiol. 1988;26(11):2257-2261). Similar responses have been seen in humans to thecapsular polysaccharides of other pathogens, and in certain instances(Pace D, Vaccine. 2009; 27 Suppl 2:B30-41; Gonzalez-Fernandez A,Vaccine. 17 2008; 26(3):292-300) progressively diminished boostresponses have been noted after sequential re-immunizations due todepletion of pre-committed naive B-cells (Richmond P, J Infect Dis.2000; 181(2):761-764). Polysaccharides are thymus-independent antigensthat do not activate T-cells and hence generally generate only moderateantibody titers without immunologic memory, class-switching, or affinitymaturation (Pollard A J, Nat Rev Immunol. 2009; 9(3):213-220).Furthermore, whereas some polysaccharides elicit acceptable antibodylevels, other polysaccharides are not immunogenic as purified antigens.Covalent chemical linkage of bacterial polysaccharides with proteins hasenhanced the magnitude, quality and duration of the induced antibody,through activation of polysaccharide-specific B-cells by protein carrierspecific helper T-cells, and importantly, has generated anamnestic andbooster responses. Glycoconjugate vaccines are among the most costly ofall vaccine types to manufacture, however, and development ofmultivalent conjugate formulations with >7 components (e.g.,pneumococcal CPS conjugates) have been hampered by issues of epitopicsuppression and interference among individual components (Dagan R,Vaccine. 2010; 28(34):5513-5523).

Since antibodies to the OPS of Klebsiella pneumoniae are protective, andthe overall number and predominance of OPS types is relatively limited,it raises the possibility that a Klebsiella pneumoniae OPS vaccineapproach might be a more straightforward and feasible vaccine strategyfor KP. Accordingly, there has been extensive investigation over theprevious decades towards vaccine strategies targeting KP LPS. Vaccineformulations utilizing whole-cell killed organisms and purified LPS,however, are unacceptably reactogenic for humans, as they elicit severeadverse reactions including high fever and malaise.

The lipid A endotoxin portion of LPS is readily cleaved by chemicalmeans, yielding isolated O polysaccharide (OPS) or a coreoligosaccharide and an O polysaccharide (COPS)(Wang X, Subcell Biochem.2010; 53:27-51; Simon R, Infect Immun. 2011; 79(10):4240-4249). Aspurified polysaccharide antigens, COPS molecules have generally provenentirely refractory to antibody production in animal models (Simon R,Infect Immun. 2011; 79(10):4240-4249; Konadu E, Infect Immun. 1996;64(7):2709-2715; Watson D C, Infect Immun. 1992; 60(11):4679-4686).However, conjugation with carrier proteins (e.g., CRM₁₉₇, flagellins,porins, tetanus toxoid [TT]) has enhanced immunogenicity (Knuf M,Vaccine. 2011; 29(31):4881-4890). COPS-based conjugate vaccines haveproven efficacious in animal models for several GNB pathogens (e.g., E.coli (Cryz S J, Jr., Infect Immun. 1990; 58(2):373-377; Konadu E, InfectImmun. 1994; 62(11):5048-5054), V. cholerae, PA (Cryz S J, Jr., InfectImmun. 1986; 52(1):161-165), Salmonella (Simon R, Infect Immun. 2011;79(10):4240-4249; Konadu E, Infect Immun. 1996; 64(7):2709-2715; WatsonD C, Infect Immun. 1992; 60(11):4679-4686; Svenson S B, Infect Immun.1979; 25(3):863-872; Micoli F, PLoS One. 2012; 7(11):e47039), Shigella(Kubler-Kielb J, Carbohydr Res. 2010; 345(11):1600-1608; Robbins J B,Proc Natl Acad Sci USA. 2009; 106(19):7974-7978; Chu C Y, Infect Immun.1991; 59(12):4450-4458)). Importantly, COPS conjugates have beenwell-tolerated and immunogenic in human clinical trials (Passwell J H,Infect Immun. 2001; 69(3):1351-1357; Cohen D, Infect Immun. 1996;64(10):4074-4077; Konadu E Y, Infect Immun. 2000; 68(3):1529-1534;Konadu E Y, J Infect Dis. 1998; 177(2):383-387; Cryz S J, Jr., J ClinInvest. 1987; 80(1):51-56; Cryz S J, Jr., J Infect Dis. 1986;154(4):682-688) and have induced functional bactericidal antibodies(Konadu E Y, Infect Immun. 2000; 68(3):1529-1534). Some COPS conjugateshave demonstrated efficacy in controlled field trials. In a largerandomized double-blind efficacy trial of a Shigella sonnei COPSconjugate among military recruits in Israel, significant protection wasobserved, for which levels of anti-S. sonnei LPS correlated withprotection (Cohen D, Lancet. 1997; 349(9046):155-159). A Pseudomonasaeruginosa COPS-based conjugate vaccine was immunogenic whenadministered to acute trauma patients within 72 hours of hospitalization(Campbell W N, Clin Infect Dis. 1996; 23(1):179-181).

All pathogenic Pseudomonas aeruginosa express a single polar flagellumthat extends from the cell surface (FIG. 2; adapted from Dasgupta N, JBacteriol. 2000; 182(2):357-364) to enable motility, that is comprisedchiefly by polymers of either type A or B flagellin proteins(Stanislaysky E S, FEMS Microbiol Rev. 1997; 21(3):243-277). There is asingle B-type flagellin form (FlaB)(Verma A et al., J Bacteriol. 1998;180(12):3209-3217), and two A-type flagellin sub-forms (FlaA) thatdiffer in sequence by only a few amino acids and are similarly reactivewith A-type specific antibodies (Brimer C D, Montie T C, J Bacteriol.1998; 180(12):3209-3217; Arora S K et al., J Bacteriol. 2004;186(7):2115-2122). While there have been no rigorous surveys conductedto determine the precise prevalence of strains expressing A and B typeflagellin, the distribution of A and B type flagella expressing clinicalisolates reported in the literature suggests that the prevalence of thetwo flagella types does not differ greatly (Rosok M J et al., InfectImmun. 1990; 58(12):3819-3828; Shanks K K et al., Clin Vaccine Immunol.2010; 17(8):1196-1202).

Pseudomonas aeruginosa flagella are well established as virulencefactors and protective antigens against Pseudomonas aeruginosainfections. The requirement of flagella for Pseudomas aeruginosapathogenicity is underscored by the dramatically reduced virulenceobserved for strains lacking flagella in mouse models of fatal Pseudomasaeruginosa wound and respiratory infections (Montie T C et al., InfectImmun. 1982; 38(3):1296-1298; Feldman M et al., Infect Immun. 1998;66(1):43-51). Several roles have been noted for flagella in Pseudomonasaeruginosa pathogenesis. Flagellar mediated motility is important forbiofilm development, and strains lacking functional motile flagella donot establish robust biofilms in vitro and are attenuated in vivo(Klausen M et al., Mol Microbiol. 2003; 48(6):1511-1524; O'Toole G A etal., Mol Microbiol. 1998; 30(2):295-304; Arora S K et al., Infect Immun.2005; 73(7):4395-4398). Accordingly, highly motile strains are extremelypathogenic in a mouse model of Pseudomas aeruginosa burn infection(Craven R C et al., Can J Microbiol. 1981; 27(4):458-460). Flagella havealso been found as attachment and colonization factors binding tomammalian epithelial cell glycans (Arora S K et al., Infect Immun. 1998;66(3): 1000-1007; Arora S K et al., Infect Immun. 1996; 64(6):2130-2136;Lu W et al., J Immunol. 2006; 176(7):3890-3894). Binding to mammalianToll-like receptor 5 (TLRS) protein by Pseudomonas flagellin activatesputative protective pro-inflammatory signaling pathways, however, overtinflammation due to flagellin is likely to be detrimental to the host (Balloy V et al., J Infect Dis. 2007; 196(2):289-296; Ben Mohamed F etal., PLoS One. 2012; 7(7):e39888).

Antibodies specific for Pseudomas aeruginosa flagellins elicited byactive immunization, or supplied by passive transfer have conferredrobust protection in animal models against respiratory (Campodonico V Let al., Infect Immun. 2011; 79(8):3455-3464; Campodonico V L et al.,Infect Immun. 2010; 78(2):746-755), peritonitis (Neville L F et al., IntJ Mol Med. 2005; 16(1):165-171) or burn wound (Faezi S et al., APMIS.2013; Barnea Y et al., Burns. 2009; 35(3):390-396; Barnea Y et al.,Plast Reconstr Surg. 2006; 117 (7): 2284-2291; Holder I A et al., JTrauma. 1986; 26(2):118-122; Holder I A et al., Am J Med. 1984;76(3A):161-167; Holder I A et al., Infect Immun. 1982; 35(1):276-280)Pseudomas aeruginosa infections. The presumed mechanism of protection byanti-Fla antibodies is arrest of motility and enhancement ofopsonophagocytic killing (Stanislaysky E S, FEMS Microbiol Rev. 1997;21(3):243-277; Doring G etal., Vaccine. 2008; 26(8):1011-1024; Faezi Setal., Burns. 2011; 37(5):865-872). Protection, including for burn woundinfections, has been found as specific for either A or B type flagellins(Holder I A et al., Infect Immun. 1982; 35(1):276-280; Montie T C etal., Infect Immun. 1982; 35(1):281-288). Mice immunized with bivalentpreparations of type A and B flagellins purified from Pseudomasaeruginosa were protected against fatal infection in the burn-sepsismodel of Pseudomas aeruginosa infection with both subtypes of flagellinexpressing strains, indicating that a broadly protective bivalentPseudomonas aeruginosa flagellin vaccine is feasible (Holder I A et al.,J Trauma. 1986; 26(2):118-122; Holder I A et al., Infect Immun. 1982;35(1):276-280). Several groups have reported robust protection againstwound infections including for MDR-Pseudomonas aeruginosa by passivetransfer of anti-flagellin polyclonal sera (Faezi S et al., Burns. 2011;37(5):865-872; Drake D et al., Can J Microbiol. 1987; 33(9):755-763), aswell as monoclonal antibodies directed against type specific FlaA andFlaB epitopes (Rosok M J et al., Infect Immun. 1990; 58(12):3819-3828;Barnea Y et al., Burns. 2009; 35(3):390-396; Barnea Y et al., PlastReconstr Surg. 2006; 117(7):2284-2291; Adawi A et al., Int J Mol Med.2012; 30(3):455-464). In one study, passive transfer of a monoclonalanti-FlaA produced equivalent survival against PA infection in burnedmice as antibiotic (imipenem) treatment (Barnea Y et al., Burns. 2009;35(3):390-396). Pseudomas aeruginosa flagellin vaccines have also beeninvestigated in human clinical trials, and were found to be welltolerated and immunogenic (Doring G et al., Proc Natl Acad Sci USA. 262007; 104(26):11020-11025; Doring G, Dorner F., Behring Inst Mitt. 1997;(98):338-344; Doring G et al., Am J Respir Crit Care Med. 1995;151(4):983-985). A double-blind randomized Phase 3 trial in cysticfibrosis patients with a bivalent Pseudomas aeruginosa A/B flagellinvaccine revealed robust and durable antibody titers, and statisticallysignificant protection (Doring G et al., Proc Natl Acad Sci USA. 262007; 104(26): 11020-11025).

Pseudomas aeruginosa flagellins are not expressed at high levelsnatively, and hence high yield expression systems are required toestablish feasibility for large-scale production. Pseudomas aeruginosaflagellins are readily expressed and purified from heterologous GramNegative Bacteria (GNB) expression systems, including Salmonella andEscherichia coli (Campodonico V L et al., Infect Immun. 2011;79(8):3455-3464; Kelly-Wintenberg K et al., J Bacteriol. 1989;171(11):6357-6362; Inaba S et al., Biopolymers. 2013; 99(1):63-72). TheFliD capping protein is an essential factor for polymerization ofsecreted flagellin monomers into flagella polymers, and in the absenceof effective FliD function, flagellin monomers are secreted into theextracellular space in an unpolymerized form. The FliD protein of E.coli is an effective substitute for Pseudomas aeruginosa FliD, andexpression of PA flagellins in E. coli leads to fully formed andfunctional flagella. By comparison, Salmonella FliD does not mediatefunctional polymerization of Pseudomonas aeruginosa flagellins intoflagella, and expression of Pseudomonas aeruginosa flagellins inSalmonella causes secretion into the cell supernatant (Inaba S et al.,Biopolymers. 2013; 99(1):63-72). It has also been shown that Pseudomonasaeruginosa flagellin expressed in a heterologous GNB system isprotective, as immunization with recombinant A-type flagellin producedin E. coli provided robust protection against burn wound infection withflagellin type A expressing Pseudomonas aeruginosa, including clinicalisolates (Faezi S et al., APMIS. 2013). Monoclonal antibodies towardsFlaA or FlaB, that have protected against burn wounds with thehomologous Fla expressing Pseudomonas aeruginosa, recognize equivalentlythe cell-associated flagellin on Pseudomas aeruginosa and therecombinant soluble Pseudomas aeruginosa flagellin expressed in E. coli(Barnea Y et al., Burns. 2009; 35(3):390-396; Adawi A et al., Int J MolMed. 2012; 30(3):455-464).

Immune responses towards the flagellins of several bacterial pathogens(e.g., Salmonella (Simon R, Infect Immun. 2011; 79(10):4240-4249;McSorley S J et al., J Immunol. 2000; 164(2):986-993), Pseudomasaeruginosa (Doring G et al., Vaccine. 2008; 26(8):1011-1024),Burkholderia (Brett P J et al., Infect Immun. 1996; 64(7):2824-2828))have provided protection in animal models against infection. Flagellinshave also been explored as carrier proteins for homologous pathogenbacterial surface polysaccharides. A conjugate vaccine comprised ofBurkholderia pseudomallei COPS with the homologous strain flagellin(FliC) enhanced the anti-polysaccharide immune response, and antibodiesinduced by this vaccine imparted robust protection against B.pseudomallei infection (Brett P J et al., Infect Immun. 1996;64(7):2824-2828). The inventors have found that conjugation ofSalmonella enterica serovar Enteritidis COPS with S. Enteritidisflagellin enhances the anti-polysaccharide immune response and protectsagainst fatal S. Enteritidis infection in mice (Simon R, Infect Immun.2011; 79(10):4240-4249; Raphael Simon J Y W et al., PLOS ONE. 2013;8(5):e64680). Conjugation of Pseudomas aeruginosa alginatepolysaccharide with a recombinant A-type Pseudomas aeruginosa flagellinwas also found to increase anti-alginate antibody levels, and elicitantibodies that protected by passive transfer against pneumonic PAinfection with both mucoid and non-mucoid strains (Campodonico V L etal., Infect Immun. 2011; 79(8):3455-3464). Importantly, in all cases,antibody levels to polysaccharide conjugated flagellin were robust andequivalent to unconjugated flagellin, indicating that conjugation doesnot interfere with anti-flagellin immunity.

There remains a need for a broad spectrum vaccine that is effectiveagainst Klebsiella pneumoniae and Pseudomonas aeruginosa. The presentinvention provides multivalent conjugates directed against variousKlebsiella pneumoniae serovars as well as Pseudomas aeruginosa for usein vaccines.

This background information is provided for informational purposes only.No admission is necessarily intended, nor should it be construed, thatany of the preceding information constitutes prior art against thepresent invention.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description ofthe embodiments and the following detailed description are exemplary,and thus do not restrict the scope of the embodiments.

According to non-limiting example embodiments, in one aspect, theinvention is directed to a single conjugate vaccine for preventingbacterial infections in a human subject caused by Klebsiella andPseudomonas bacteria wherein the conjugate vaccine is comprised offlagellin proteins of Pseudomonas and surface polysaccharide antigensand/or the core oligosaccharides of Klebsiella.

In one embodiment the present invention relates to a single conjugatevaccine for preventing bacterial infections in a human subject caused byKlebsiella and Pseudomonas bacteria wherein the conjugate vaccine iscomprised of flagellin proteins or fragments or derivatives thereof ofPseudomonas and O polysaccharide antigens and/or the coreoligosaccharides derived from Klebsiella.

In another embodiment the present invention relates to a conjugatevaccine composition comprising six individual antigens selected from twoflagellin proteins or fragments or derivatives thereof derived fromPseudomonas species and nine O polysaccharide (OPS) antigens selectedfrom Klebsiella species.

In another embodiment the present invention relates to a singlequadrivalent conjugate vaccine comprising two Pseudomonas speciesflagellins or fragments or derivatives thereof as carriers for fourKlebsiella species O polysaccharide antigens.

In another embodiment the present invention relates to a method forpreparing a conjugate vaccine for preventing Pseudomonas and Klebsiellabacterial infections comprising linking OPS antigens and flagellinproteins or fragments or derivatives thereof using a variety of chemicalcrosslinking agents.

In another embodiment the present invention relates to a method forpreparing a conjugate vaccine for preventing Pseudomonas and Klebsiellabacterial infections comprising expressing the bacterial flagellinproteins of Pseudomonas in a variety of suitable bacterial expressionvectors and purifying the OPS from Klebsiella using a variety ofcommonly used methods followed by crosslinking of the OPS antigens withthe flagellin proteins.

In another embodiment the present invention relates to a passiveimmunization method for treating a human subject with a Pseudomonas orKlebsiella bacterial infection with immunologically effective amount ofan intravenous immunoglobulin preparation (IVIG) prepared from a humanhost which has been vaccinated with a conjugate vaccine comprising Opolysaccharides or core oligosaccharides from Klebsiella with flagellinproteins or fragments or derivatives thereof.

In another embodiment the present invention relates to a method foreliciting a passive immune response in a subject comprisingadministering to the subject in need thereof an immunologicallyeffective amount of an intravenous immunoglobulin preparation preparedby administering to animals a conjugate vaccine comprising an Opolysaccharide (OPS) from a Klebsiella species covalently linked to aflagellin protein or fragment or a derivative thereof from a Pseudomonasspecies.

In another embodiment the present invention relates to a method forconstructing a conjugate vaccine for eliciting an immune response in asubject in need thereof comprising producing recombinant microorganismswhich produce large amounts of Pseudomas aeruginosa flagellin orfragments or derivatives thereof and Klebsiella pneumoniae Opolysaccharides.

In another embodiment the present invention relates to a method forproducing a conjugate vaccine for Klebsiella and/or Pseudomonasinfections comprising producing recombinant microorganisms which producelarge amounts of Pseudomonas bacterial flagellins or fragments orderivatives thereof which are then conjugated (linked) with Opolysaccharides or core oligosaccharides from recombinant Klebsiellabacterial strains wherein capsule removal from the Klebsiella strain isnot required.

In another embodiment the present invention relates to a method forproducing a conjugate vaccine for Klebsiella and/or Pseudomonasinfections comprising producing recombinant bacterial expression systemsusing E. coli, Salmonella, or Pseudomonas which are engineered toproduce large amounts of bacterial flagellins or fragments orderivatives thereof into culture supernatant which are then conjugated(linked) with O polysaccharides or core oligosaccharides produced fromrecombinant attenuated Klebsiella bacterial strains.

In another embodiment the present invention relates to a method foreliciting an active immune response and antibody production in a subjectcomprising administering to the subject in need thereof animmunologically effective amount of a conjugate vaccine comprising an Opolysaccharide (OPS) from a Klebsiella species covalently linked to aflagellin protein or fragment or a derivative thereof from a Pseudomonasspecies wherein the flagellin protein acts as a carrier protein(s) inthe conjugate vaccine.

In another embodiment the present invention relates to a method forinducing an immune response in a mammal comprising administering to thesubject in need thereof a conjugate vaccine comprising an Opolysaccharide (OPS) from a Klebsiella species covalently linked to aflagellin protein or fragment or a derivative thereof from a Pseudomonasspecies wherein the dosage of vaccine is about 5 to about 50 micrograms.

In another embodiment the present invention relates to a method forinducing an immune response in a mammal comprising administering to thesubject in need thereof a conjugate vaccine comprising an Opolysaccharide (OPS) from a Klebsiella species covalently linked to aflagellin protein or fragment or a derivative thereof from a Pseudomonasspecies wherein the wherein the route of administration is subcutaneous,intravenous, intradermal, intramuscular or intranasal.

In another embodiment the present invention relates to a method forinducing an immune response in a mammal comprising administering to thesubject in need thereof a conjugate vaccine comprising an Opolysaccharide (OPS) from a Klebsiella species covalently linked to aflagellin protein or fragment or a derivative thereof from a Pseudomonasspecies along with an adjuvant selected from the group comprising orconsisting of alum, a PRR ligand, TLR3 ligand, TLR4 ligand, TLRS ligand,TLR6 ligand, TLR7/8 ligand, TLR9 ligand, NOD2 ligand, and lipid A andanalogues thereof.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1. Sequence of selected KP OPS serotypes and core polysaccharide.The OPS repeat structures for the 4 vaccine serotypes proposed areshown; CP primer is the terminal attachment point to the common Core PS(adapted from Vinogradov E et al., J Biol Chem.; 277(28):25070-25081).

FIG. 2. Transmission electron micrograph of PA strain PAK (Type Aflagella) adapted from Dasgupta et al.

FIG. 3. Salmonella with deletions in clpP or clpX are hyperflagellated.A) Electron micrograph of S. Paratyphi A; B) Motility of recombinant S.Enteritidis strains and C) Coomassie-stained SDS-PAGE and western blot(anti-FliC) of recombinant S. Enteritidis strains. Lanes 1, S.Enteritidis R11; 2, CVD 1940 (R11 ΔguaBA); 3, R11 ΔclpP; 4, CVD 1941(R11 ΔguaBA ΔclpP); 5, R11 ΔguaBA ΔclpX; 6, CVD 1942 (R11 ΔguaBA ΔfliD);7, CVD 1943 (R11 ΔguaBA ΔclpP ΔfliD).

FIG. 4. Purification of FliC and COPS from attenuated CVD 1943 S.Enteritidis. A. OD₆₀₀ versus time for two independent 20 L scalefermentor runs of CVD 1943; B. Protein accumulation in fermentationsupernatant; C. HPLC-SEC (SDS-PAGE inset) of final CVD 1943 FliCpurified from fermentation supernatant; D. SDS-PAGE with polysaccharidestain on LPS from fermentation culture (lane 1=LPS standards, 2=CVD 1943cells); E. HPLC-SEC of purified CVD 1943 COPS polysaccharide(Red=Refractive Index, Black=Abs 252 nm); F. HPAEC-PAD monosaccharidecomposition analysis for CVD 1943 COPS (indicated peaks based onmonosaccharide standards).

FIG. 5. Conjugation of S. Enteritidis COPS conjugates. (A and B) Four to20% SDS-PAGE showing Coomassie blue (A) or Pro-Q (B) staining of COPSconjugates. Lanes 1, protein standards; 2, 10 μg S. Enteritidis LPS; 3,10 μg S. Enteritidis flagella; 4, 10 μg COPS:FliC CDAP linked conjugate;5, 10 μg COPS:FliC oxime linked conjugate.

FIG. 6. Humoral immune responses in BALB/c mice following immunizationwith PBS sham, S. Enteritidis COPS, COPS admixed with flagellinmonomers, or O:H 1:1 lot 1 conjugate. Serum anti-LPS IgG (A) andanti-flagellin IgG (B) titers in individual mice (∘) and geometric means() before immunization (time 0) and at 21 days following the 1st (1),2nd (2), or 3rd (3) immunization. “*” compared to PBS by Mann-WhitneyRank-Sum test.

FIG. 7. Western blot with COPS:FliC sera: Pooled sera from miceimmunized with PBS or S. Enteritidis COPS:FliC was used to probe (1) S.Enteritidis R11 lysate, (2) R11 flagellin mutant (AFliC) lysate, (3)purified S. Enteritidis FliC, and (4) purified S. Enteritidis LPS.

FIG. 8. Immunogold labeling of S. Enteritidis strain S15. Flagellalabeled with sera from mice immunized with S. Enteritidis flagellin.Bar, 500 nm.

FIG. 9. Opsonophagocytic uptake of wild-type S. Enteritidis R11 by J774mouse macrophages exposed to sera from mice immunized with COPSconjugates. Uptake of wild-type S. Enteritidis R11 and derivativesmutated in invA, fliC, and rfaL in the presence of pooled sera from miceimmunized with COPS:FliC conjugate relative to sera from mice receivingPBS.

FIG. 10. Flow cytometric assay for the uptake of PA by humanpolymorphonuclear leukocytes (PMNs). GFP-expressing strain PAO1 wasadded to PMNs (MOI=5) in the presence (blue line) or absence (red line)of IVIG enriched in antibodies to PA and KP (Cryz S J et al., J InfectDis. 1991; 163(5):1055-1061). PA were spun onto PMNs at 4° C., washed,and incubated for 15 min at 37° C. PMNs were then washed, incubated for15 min in gentamicin (50 μg/ml), washed and resuspended for analysis.

FIG. 11. Endpoint OPA titers for mice immunized with live attenuated S.Typhimurium CVD 1931. Line indicates mean titers. I, Immune; NI,Non-immune serum. ****, P<0.0001 by Mann-Whitney test.

FIG. 12. Schematic diagram of the guaBA and wzabc genes in Klebsiellapneumoniae subsp. pneumoniae MGH 78578, GenBank: CP000647.1

FIG. 13. Schematic diagram of the guaBA and wzabc gene deletions. Thegray boxes represent the portion of genome remaining, the blue linkerrepresents the portion of genome deleted.

FIG. 14. Confirmation of deletion of guaBA by PCR. Lane 1, Kp B5055(O1:K2) wild type; 2, Kp B5055 AguaBA; 3, Kp 390 (O3:K11) wild type; 4,Kp 390 AguaBA; 5, Kp 7380 (O2:K-) wild type; 6, Kp 7380 AguaBA. Primerpairs used for the amplification: lanes 1 and 2,guaBA_256_F+guaBA_155_R; lanes 3 to 6, guaB_F2+guaA_R2.

FIG. 15. Test for guanine auxotrophy of K. pneumoniae AguaBA reagentstrains. CDM=chemically defined medium; CDM+guanine=CDM supplementedwith 0.1% guanine.

FIG. 16. Schematic diagram of pSEC10 containing fliC from P. aeruginosaPAK (pSEC10-flaA).

FIG. 17. Schematic diagram of pSEC10 containing fliC from P. aeruginosaPAO1 (pSEC10-flaB).

FIG. 18. Schematic diagram showing BLAST analysis of the sequenced scarregion against S. Enteritidis. The gray boxes (Query) are the sequencesremaining in CVD 1947. The blue linker is what has been deleted from CVD1943. AV78_06120=fliC.

FIG. 19. Expression and secretion of rFlaA and rFlaB in CVD 1947.SDS-PAGE and coomassie analysis of cell pellets (lanes 1, 3) andsupernatants (lanes 2, 4) from liquid cultures of CVD1947-pSEC10_flaA(lanes 1, 2) and CVD 1947-pSEC10_flaB (lanes 3, 4) grown in Hy-Soy.Lane: M, molecular weight standards.

FIG. 20. Reactivity of recombinant P. aeruginosa flagellin FlaA secretedfrom S. Enteritidis CVD 1947 by sera raised against native FlaA. Cells(lane 1) and supernatants (lane 2) from liquid growth cultures ofCVD1947-pSEC10_flaA were assessed by Western blot with polyclonal serafrom mice immunized with FlaA purified from PAK

FIG. 21. O1 OPS purification after release by acetic acid/100° C.In-process and purified material was assessed by HPLC-SEC through aBiosep SEC4000 column at lml/minute in PBS with detection by RefractiveIndex. Chromatogram trace: Grey, 30 kDa TFF retentate; black,post-hydrolysis supernatant; blue, anion-exchange flow-through;red/aqua, 10 kDa TFF permeate; pink, 10 kDa TFF retentate.

FIG. 22. O1 OPS purification after release by nitrous acid deamination.In-process and purified material was assessed by HPLC-SEC through aBiosep SEC4000 column at lml/minute in PBS with detection by RefractiveIndex. Chromatogram trace: Black, 30 kDa TFF retentate; grey,post-hydrolysis supernatant; blue, 10 kDa TFF retentate.

FIG. 23. HPAEC-PAD analysis of depolymerized purified O1 OPS obtained byacid/heat hydrolysis. Samples were passed through a Carbopac PA10 at0.010 ml/minute in 18 mM KOH. Saccharide peaks were identified usingcommercially available monosaccharide standards.

FIG. 24. Purification of P. aeruginosa flagellin produced in CVD1947.Purified fraction (lane 1) was assessed for size and purity by SDS-PAGEwith coomassie staining Lane: M, molecular weight standards; 2, purifiedrFlaA.

FIG. 25. HPLC-SEC analysis of conjugated and unconjugated KP-O1 OPS andrPA-FlaA. Chromatogram for Purified KP-O1 OPS (Black line), rPA-FlaA(grey line), and CDAP linked KP-O1:rPA-FlaA conjugate (blue line)analyzed by HPLC-SEC through a Biosep SEC4000 column at lml/min in PBSwith detection by Refractive index.

FIG. 26. SDS-PAGE analysis of rFlaA and KP-O1:rPA-FlaA conjugate.Coomassie staining (A) and Western blot with polyclonal mouse anti-FlaAsera (B). Sample and protein amount run are detailed in the figure foreach lane.

FIG. 27. Dot blot analysis for KP-O1 OPS and KP-O1:rPA-FlaA withanti-KP-O1 sera. Equivalent amounts of polysaccharide either asconjugate with rFlaA or alone were blotted onto a PVDF membrane andprobed with polyclonal mouse sera raised against CVD 3001 (KP-O1 OPS).

FIG. 28. Diagram of challenge study. Sun-type KP-O1-OPS:PA-FlaAconjugate (oxime), lattice type conjugate (CDAP), O1 OPS alone oradmixed with FlaA will be used to immunize mice (black arrow). PBS iscontrol. Levels of vaccine induced IgG antibodies in sera beforeimmunization and 21 days after each vaccine dose will be measured byELISA, motility inhibition and OPA assays (red arrow). Mice will bechallenged with IP with KP or in burn wound infections with PA. Forchallenge, we will use PA PAK (type A flagellin-expressing isolate) or(KP B5055).

FIG. 29. Diagram of challenge study. The most effectiveKP-O1-OPS:PA-FlaA conjugate and O1 OPS admixed with FlaA will be used toimmunize mice (black arrow). PBS is control. Levels of vaccine inducedIgG antibodies in sera before immunization and 21 days after eachvaccine dose will be measured by ELISA, motility inhibition and OPAassays (red arrow). Mice will be challenged with burn, myositis, punchwound, or IP septicemia PA infection routes. We will use PA PAK (type Aflagellin-expressing isolate) or (KP B5055).

FIG. 30. Diagram of challenge study. Screen for functional efficacy ofvaccine-elicited antibodies in vivo by measuring protection against IPinfection with the homologous KP O type expressing strain (O1: B5055;O2, O3 and O5: recombinant mouse virulent strains), and burn wounds withthe homologous flagellin expressing PA strain. Black arrow isintroduction of immunogen. Red arrow is blood sampling to measure levelsof vaccine induced IgG antibodies in sera before immunization and 21days after each vaccine dose by ELISA, motility inhibition and OPAassays.

FIG. 31. Diagram of challenge study. To confirm that the specific immuneresponses to FlaA and FlaB are maintained when co-formulated, mice willbe immunized 3 times at 28 day intervals with monovalent and bivalentflagellin preparations (black arrow). Levels of IgG and functionaltiters for the homologous Ha types will be determined in pre-immune seraand sera taken 21 days after the final dose (red arrow). Protection willbe assessed against burn infection with homologous Fla expressing PAstrains.

FIG. 32. Diagram of challenge study. An assay will be conducted toconfirm that the humoral responses and protective efficacy of the 4down-selected monovalent COPS and flagellin conjugate vaccine componentsare maintained when administered as a multivalent vaccine formulation.Mice will be immunized 3 times at 28 day intervals with PBS or thequadrivalent formulation, or 2 individual monovalent conjugates (blackarrow). Sera obtained prior to immunization and 21 days after the finaldose will be assessed for anti-LPS and anti-flagellin antibodies (redarrow). The protective efficacy of quadrivalent—relative tomonovalent—vaccines to prevent invasive and wound infections will bedetermined using the IP, myositis, burn wound or punch-biopsy models andhomologous KP O-type pathogens or the homologous flagellin typeexpressing PA (PAK or PA:O1).

FIG. 33. Diagram of challenge study. We will assess the utility of thequadrivalent conjugate formulation to generate antibody preparations.Rabbits will be hyper-immunized with quadrivalent vaccine and pooledsera will be prepared for use in passive transfer studies in mice. Thelevel of anti-LPS and anti-flagellin IgG in rabbit sera will bedetermined by ELISA, OPA and motility inhibition assays. Dosage levelswill be approximated to the antibody titer induced by activeimmunization in mice. Naive mice will be intravenously administeredimmune sera, normal (unimmunized) rabbit sera (N.S.), or PBS, followedby IP or burn infection 2-4 hours later with KP B5055 or PA PAK,respectively.

FIG. 34. Diagram of challenge study. The 50% effective dose (ED5₀) forvarious doses of KP B5055 O1:K2 and PA PAK will be determined byinfecting at various doses at multiple wound sites in naive pigs. Once areliable infectious dose has been determined, we will immunize 4 pigs 3times with PBS or quadrivalent conjugate containing 25 μg of totalpolysaccharide (black arrow). As controls, 2 pigs will be mock immunizedwith PBS alone. Twenty-one days after the final dose, immunized orcontrol pigs will be infected at multiple sites with moderate or highlevels of KP B5055 of PA PAK. Red arrows indicate blood sampling forantibody assays.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a conjugate vaccine for Klebsiella andPseudomonas bacterial infections. These bacteria are known to cause awide variety of infections in human subjects including but not limitedto wound infections, burn infections, urinary tract infections,respiratory infections, central nervous system infections, abscessformations, cystic fibrosis, in-dwelling catheter infections, invasivebacteremia, and septic shock. There is a real need to develop vaccineswhich can protect against such bacterial infections. In someembodiments, the present invention relates to a vaccine product whichencompasses a tetravalent formulation of four different OPSpolysaccharide antigens from Klebsiella pneumoniae serovars conjugatedwith two different flagellin proteins from Pseudomonas aeruginosa. Thevaccine can have efficacy for therapeutic use to mitigate againstmultiple drug resistant Pseudomonas and Klebsiella bacterial infections.

At present, there is no simple and broadly effective vaccine which iseffective against both Klebsiella and Pseudomonas. In some aspects, theinvention described herein is a novel conjugate vaccine which comprisesantigens from both bacterial types and can be manufactured in a largescalable fashion. Moreover, in some embodiments, the vaccine could alsobe used to generate therapeutic immunoglobulin (IVIG) preparations forpassive protection against acute infections.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN O19879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

For the purpose of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). The use of “or” means“and/or” unless stated otherwise. The use of “a” herein means “one ormore” unless stated otherwise or where the use of “one or more” isclearly inappropriate. The use of “comprise,” “comprises,” “comprising,”“include,” “includes,” and “including” are interchangeable and notintended to be limiting. Furthermore, where the description of one ormore embodiments uses the term “comprising,” those skilled in the artwould understand that, in some specific instances, the embodiment orembodiments can be alternatively described using the language“consisting essentially of' and/or “consisting of.”

Conjugate

In one aspect, the present invention is directed to a conjugatecomprising a Klebsiella surface polysaccharide antigen and a Pseudomonasflagellin protein or antigenic fragment or derivative thereof. Inparticular aspects of the invention, the surface polysaccharide antigenand the flagellin or antigenic fragment or derivative thereof arecovalently linked optionally via a linker.

The Klebsiella surface polysaccharide antigen can be any knownKlebsiella surface polysaccharide antigen or a derivative or antigenicfragment thereof. In some embodiments, the surface polysaccharide isfrom one or more Klebsiella pneumoniae serovars. In some aspects of theinvention, the Klebsiella surface polysaccharide antigen can be an Opolysaccharide (OPS), a core oligosaccharide and an O polysaccharide(COPS), a capsule polysaccharide or combinations thereof.

As used herein, “OPS” is a polysaccharide in which the lipid A moietyfrom lipopolysaccharide (LPS) and core oligosaccharide have beenremoved. In some embodiments of the invention, the surfacepolysaccharide antigen is an OPS. In some embodiments, the surfacepolysaccharide antigen is from epidemiologically relevant Klebsiella Oserovars such as Klebsiella pneumoniae serovar O1, O2a, O3 and O5. Insome embodiments, the surface polysaccharide antigen is an OPS derivedfrom Klebsiella pneumoniae serovars O1, O2a, O2a,c, O3, O4, O5, O7, O8and O12. In some embodiments, the surface polysaccharide antigen is anOPS derived from Klebsiella pneumoniae serovars O1, O2a, O3 and O5.

The Pseudomonas flagellin can be any known Pseudomonas flagellin. Asused herein, the term “flagellin” encompasses flagellin, fragments offlagellin and derivatives thereof. In particular aspects of theinvention, the Pseudomonas flagellin is a Pseudomas aeruginosa (PA)flagellin. It is believed that all pathogenic Pseudomas aeruginosaexpress a single polar flagellum that extends from the cell surface toenable motility, that is comprised chiefly by polymers of either type Aor B flagellin proteins. In some aspects of the invention, thePseudomonas flagellin is a Pseudomas aeruginosa (PA) flagellin type A(FlaA) or an antigenic fragment or derivative thereof and/or a Pseudomasaeruginosa flagellin type B (FlaB) or an antigenic fragment orderivative thereof. In some embodiments, the Pseudomonas aeruginosaflagellin type A (FlaA) comprises SEQ ID NO:1 or an antigenic fragmentor derivative thereof. In some embodiments, the Pseudomas aeruginosaflagellin type B (FlaB) comprises SEQ ID NO:2 or an antigenic fragmentor derivative thereof. FliC and Fla (e.g., FlaA and FlaB) are usedinterchangeably throughout the specification but all refer to flagellin.

In some embodiments, the conjugate comprises i) Pseudomonas aeruginosaflagellin type A (FlaA) or an antigenic fragment or derivative thereofand/or Pseudomas aeruginosa flagellin type B (FlaB) or an antigenicfragment or derivative thereof and ii) OPS from Klebsiella pneumoniaeselected from Klebsiella pneumoniae serovars O1, O2a, O3, O5 orcombinations thereof. In some embodiments, Pseudomonas flagellin or anantigenic fragment or derivative thereof can be covalently linked to oneor more OPS from a single Klebsiella pneumoniae serovar type or may belinked to OPS from multiple Klebsiella pneumoniae serovar types.

The ratio or stoichiometry of surface polysaccharide to flagellin is notlimiting. In some embodiments, the Pseudomonas flagellin or an antigenicfragment or derivative thereof can be linked to 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more surface polysaccharides, such as OPS, from the sameKlebsiella or from mixtures of Klebsiella serovar types.

In some embodiments, the Pseudomonas flagellin or an antigenic fragmentor derivative thereof is linked to one to four OPS from the same serovartype. In another embodiment, the Pseudomonas flagellin or an antigenicfragment or derivative thereof is linked to one to four OPS from atleast two different serovar types. In another embodiment, the flagellinor an antigenic fragment or derivative thereof is linked to one to fourOPS, each from different serovar types. In some embodiments, theKlebsiella serovars comprise Klebsiella pneumoniae serovar O1, O2a, O3,and O5.

In one embodiment, the conjugate comprises SEQ ID NO:1 or an antigenicfragment or derivative thereof and a surface polysaccharide fromKlebsiella pneumoniae serovars O1, O2a, O3, O5 or combinations thereof.In some embodiments, the surface polysaccharide is OPS.

In another embodiment, the conjugate comprises SEQ ID NO:2 or anantigenic fragment or derivative thereof and a surface polysaccharidefrom Klebsiella pneumoniae serovars O1, O2a, O3, O5 or combinationsthereof. In some embodiments, the surface polysaccharide is OPS.

Examples of fragments or derivatives of Pseudomonas flagellin caninclude fragments of the natural protein, including internal sequencefragments of the protein that retain their ability to elicit protectiveantibodies against a desired bacteria. The derivatives are also intendedto include variants of the natural protein such as proteins havingchanges in amino acid sequence but that retain the ability to elicit animmunogenic, biological, or antigenic property as exhibited by thenatural molecule.

By derivative is further meant an amino acid sequence that is notidentical to the wild type amino acid sequence, but rather contains atleast one or more amino acid changes (deletion, substitutions,inversion, insertions, etc.) that do not essentially affect theimmunogenicity or protective antibody responses induced by the modifiedprotein as compared to a similar activity of the wild type amino acidsequence, when used for the desired purpose. In some embodiments, aderivative amino acid sequence contains at least 85-99% homology at theamino acid level to the specific amino acid sequence. In furtherembodiments, the derivative has at least 90% homology at the amino acidlevel. In other embodiments, the derivative has at least 95% homology.

The flagellin of the invention may be a peptide encoding the nativeamino acid sequence or it may be a derivative or antigenic fragment ofthe native amino acid sequence.

In some embodiments, the surface polysaccharide antigen of a Klebsiellais covalently linked to the Pseudomonas flagellin protein or anantigenic fragment or a derivative thereof either directly or with alinker. In some embodiments, the linker or linking chemical is selectedfrom 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), adipicacid dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal,D-glucuronolactone or p-nitrophenylethyl amine. In a particularembodiment, the linking chemical is CDAP.

Compositions

In some embodiments, the invention provides compositions comprising theconjugates of the invention. In some embodiments, the compositions arevaccine compositions which provide protective immunity against one ormore Klebsiella and/or Pseudomonas pathogens and which comprise one ormore of the above-mentioned conjugates. In some embodiments, effectiveamounts of one or more unconjugated Pseudomonas flagellin can be addedto the compositions of the invention. In some embodiments, adding one ormore unconjugated flagellin to compositions comprising one or moreconjugates can enhance the immune response to the flagellin epitopes. Insome embodiments, the one or more unconjugated Pseudomonas flagellin isselected from flagellin comprising SEQ ID NO:1, SEQ ID NO:2, antigenicfragments and derivatives thereof and combinations thereof.

In some embodiments of the invention, the vaccine composition is amultivalent conjugate vaccine comprising one or more Pseudomonasflagellins linked to one or more Klebsiella surface polysaccharides,such as O polysaccharides (OPS). For example, the composition can be amultivalent conjugate vaccine comprising two different Pseudomonasflagellin proteins or antigenic fragments or derivatives thereofcovalently linked to one or more Klebsiella O polysaccharides (OPS). Insome embodiments, the multivalent conjugate vaccine comprises OPSantigens from one or more of Klebsiella pneumoniae serovars O1, O2a,O2a,c, O3, O4, O5, O7, O8 and O12. In some embodiments, the multivalentconjugate vaccine comprises four different OPS antigens from Klebsiellapneumoniae serovars O1, O2a, O3, and O5. In some embodiments, thePseudomonas is Pseudomonas aeruginosa.

In some embodiments, the composition comprises an effective amount ofone or more conjugates comprising a Pseudomonas flagellin protein or anantigenic fragment or derivative thereof and a surface polysaccharidefrom Klebsiella. In one embodiment, the composition comprises aPseudomas aeruginosa flagellin or an antigenic fragment or derivativethereof and an OPS from Klebsiella pneumoniae serovars O1, O2a, O3, O5or combinations thereof.

In some embodiments, the composition comprises a multivalent conjugatevaccine comprising one or more Pseudomas aeruginosa flagellin proteinsselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 andcombinations thereof, or antigenic fragments or derivative thereof andone or more OPS from Klebsiella pneumoniae serovars O1, O2a, O3, O5 orcombinations thereof.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovars O1, O2a, O3, O5 or combinations thereof; and    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovars O1, O2a, O3, O5 or combinations thereof.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O1;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O2a;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O3; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O5.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O1;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O3;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O2a; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O5.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O1;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O5;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O2a; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and one or more OPS from Klebsiella        pneumoniae serovar O3.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O2a;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O5;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O1; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O3.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O3;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O5;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O1; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O2a.

In some embodiments, the composition comprises:

-   -   i) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O2a;    -   ii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:1 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O3;    -   iii) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O1; and    -   iv) a conjugate comprising a Pseudomas aeruginosa flagellin        protein according to SEQ ID NO:2 or an antigenic fragment or        derivative thereof and OPS from Klebsiella pneumoniae serovar        O5.

In some embodiments, the invention provides a composition comprising aneffective amount of sera from a subject administered one or moreconjugates of the invention. In some embodiments, the invention providesa composition comprising an effective amount of purified or enrichedimmunoglobulins from a subject administered one or more conjugates ofthe invention. In some embodiments, the composition comprising sera orthe immunoglobulins can be administered to a subject in immunotherapyapplications.

In some embodiments, the compositions are pharmaceutical compositionscomprising one or more conjugates of the invention and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition can contain salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. Adjuvants are substances that can be used to specificallyaugment a specific immune response. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the animal beingimmunized. Adjuvants can be loosely divided into several groups basedupon their composition. These groups include oil adjuvants (for example,Freund's complete and incomplete), mineral salts (for example,AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄ (SO₄), silica, kaolin, and carbon),polynucleotides (for example, poly IC and poly AU acids), and certainnatural substances (for example, wax D from Mycobacterium tuberculosis,as well as substances found in Corynebacterium parvum, or Bordetellapertussis, and members of the genus Brucella. Adjuvants are described byWarren et al. (Ann. Rev. Biochem., 4:369-388, 1986), the entiredisclosure of which is hereby incorporated by reference. Furtheradjuvants suitable for use in the present invention include alum, a PRRligand, TLR3 ligand, TLR4 ligand, TLR5 ligand, TLR6 ligand, TLR7/8ligand, TLR9 ligand, NOD2 ligand, and lipid A and analogues thereof.

In some embodiments of the invention the use of a flagellin protein orantigenic fragment or derivative thereof as a carrier for a conjugateprovides an inherent adjuvant boost, and stimulates a robust immuneresponse without the addition of further adjuvant. Thus, in someembodiments, the flagellin protein antigenic fragment or derivativethereof acts an adjuvant which stimulates innate immunity through TLR5to improve the immunogenicity of surface polysaccharide antigen (e.g.,OPS) within the composition. In some embodiments, the carrier is amutant flagellin antigenic fragment or derivative thereof which has adiminished capability to stimulate innate immunity through TLR5. In someembodiments, an adjuvant is added to the compositions while in otherembodiments, no adjuvant is added.

In some embodiments, conventional adjuvants can be administered. Amongthose substances that can be included are the saponins such as, forexample, Quil A. (Superfos A/S, Denmark). In some embodiments,immunogenicity of the conjugates in both mice and rabbits is enhanced bythe use of monophosphoryl lipid A plus trehalose dimycolate (Ribi-700;Ribi Immunochemical Research, Hamilton, Mont.) as an adjuvant. Alum, aPRR ligand, TLR3 ligand, TLR 4 ligand, TLR5 ligand, TLR6 ligand, TLR7/8ligand, TLR9 ligand, NOD2 ligand, and lipid A and analogues thereof mayseparately or in combination may also be used as adjuvants. Examples ofmaterials suitable for use in vaccine compositions are provided inRemington's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co,Easton, Pa., pp. 1324-1341 (1980), which disclosure is incorporatedherein by reference).

In some embodiments, the vaccine composition can be formulated intoliquid preparations for, e.g., nasal, rectal, buccal, vaginal, peroral,intragastric, mucosal, perlinqual, alveolar, gingival, olfactory, orrespiratory mucosa administration. Suitable forms for suchadministration include solutions, suspensions, emulsions, syrups, andelixirs. The vaccine composition can also be formulated for parenteral,subcutaneous, intradermal, intramuscular, intraperitoneal or intravenousadministration, injectable administration, sustained release fromimplants, or administration by eye drops. Suitable forms for suchadministration include sterile suspensions and emulsions. Such vaccinecomposition can be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose, and thelike. The vaccine composition can also be lyophilized The vaccinecomposition can contain auxiliary substances such as wetting oremulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Texts, such as Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) andRemington's Pharmaceutical Sciences, Mack Pub. Co.; 18^(th) and 19^(th)editions (December 1985, and June 1990, respectively), incorporatedherein by reference in their entirety, can be consulted to preparesuitable preparations. Such preparations can include complexing agents,metal ions, polymeric compounds such as polyacetic acid, polyglycolicacid, hydrogels, dextran, and the like, liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

In some embodiments, the vaccine composition of the invention isadministered parenterally. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. In some embodiments, the vaccine compositionfor parenteral administration may be in the form of a sterile injectablepreparation, such as a sterile injectable aqueous or nonaqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Carriers orocclusive dressings can be used to increase skin permeability andenhance antigen absorption. Suspensions may be formulated according tomethods well known in the art using suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation mayalso be a sterile injectable solution or suspension in a parenterallyacceptable diluent or solvent, such as a solution in 1,3-butanediol.Suitable diluents include, for example, water, Ringer's solution andisotonic sodium chloride solution. In addition, sterile fixed oils maybe employed conventionally as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid maylikewise be used in the preparation of injectable preparations.

Liquid dosage forms for oral administration may generally comprise aliposome solution containing the liquid dosage form. Suitable forms forsuspending liposomes include emulsions, suspensions, solutions, syrups,and elixirs containing inert diluents commonly used in the art, such aspurified water. Besides the inert diluents, such compositions can alsoinclude adjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

In some embodiments, the vaccine composition is provided as a liquidsuspension or as a freeze-dried product (or freeze-dried hyperimmuneglobulin for oral administration). Suitable liquid preparations include,e.g., isotonic aqueous solutions, suspensions, emulsions, or viscouscompositions that are buffered to a selected pH. Transdermalpreparations include lotions, gels, sprays, ointments or other suitabletechniques. If nasal or respiratory (mucosal) administration is desired(e.g., aerosol inhalation or insufflation), compositions can be in aform and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or adose having a particular particle size, as discussed below.

In some embodiments, when in the form of solutions, suspensions andgels, in some embodiments, the composition contains a major amount ofwater (preferably purified endotoxin-free water) in addition to theactive ingredient. Minor amounts of other ingredients such as pHadjusters, emulsifiers, dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, colors, and the like canalso be present.

In some embodiments, the compositions are preferably isotonic with theblood or other body fluid of the recipient. The isotonicity of thecompositions can be attained using sodium tartrate, propylene glycol orother inorganic or organic solutes. Sodium chloride is particularlypreferred. Buffering agents can be employed, such as acetic acid andsalts, citric acid and salts, boric acid and salts, and phosphoric acidand salts. In some embodiments of the invention, phosphate bufferedsaline is used for suspension.

In some embodiments, the viscosity of the compositions can be maintainedat the selected level using a pharmaceutically acceptable thickeningagent. In some embodiments, methylcellulose is used because it isreadily and economically available and is easy to work with. Othersuitable thickening agents include, for example, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and thelike. The concentration of the thickener can depend upon the agentselected. In some embodiments, viscous compositions are prepared fromsolutions by the addition of such thickening agents.

In some embodiments, a pharmaceutically acceptable preservative can beemployed to increase the shelf life of the compositions. Benzyl alcoholcan be suitable, although a variety of preservatives including, forexample, parabens, thimerosal, chlorobutanol, or benzalkonium chloridecan also be employed. A suitable concentration of the preservative canbe from 0.02% to 2% based on the total weight although there can beappreciable variation depending upon the agent selected.

In some embodiments, pulmonary delivery of the composition can also beemployed. In some embodiments, the composition is delivered to the lungsof a mammal while inhaling and traverses across the lung epitheliallining to the blood stream. A wide range of mechanical devices designedfor pulmonary delivery of therapeutic products can be employed,including but not limited to nebulizers, metered dose inhalers, andpowder inhalers, all of which are familiar to those skilled in the art.These devices employ formulations suitable for the dispensing of theconjugate. Typically, each formulation is specific to the type of deviceemployed and can involve the use of an appropriate propellant material,in addition to diluents, adjuvants and/or carriers useful in therapy.

In embodiments where the compositions are prepared for pulmonarydelivery in particulate form, it has an average particle size of from0.1 μm or less to 10 μm or more. In some embodiments, it has an averageparticle size of from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μmto about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 μm for pulmonary delivery.Pharmaceutically acceptable carriers for pulmonary delivery of theinsufflation include carbohydrates such as trehalose, mannitol, xylitol,sucrose, lactose, and sorbitol. Other ingredients for use informulations can include DPPC, DOPE, DSPC and DOPC. Natural or syntheticsurfactants can be used, including polyethylene glycol and dextrans,such as cyclodextran and other related enhancers, as well as celluloseand cellulose derivatives, and amino acids can also be used. Liposomes,microcapsules, microspheres, inclusion complexes, and other types ofcarriers can also be employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, typically comprise the composition dissolved or suspended inwater at a concentration of about 0.01 or less to 100 mg or more ofconjugate per mL of solution, preferably from about 0.1, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90 mg of conjugate per mL of solution. Theformulation can also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the conjugate or composition caused byatomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the vaccine compositionsuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such chlorofluorocarbon, ahydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons, such astrichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, andcombinations thereof. Suitable surfactants include sorbitan trioleate,soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing the vaccine composition,optionally including a bulking agent, such as lactose, sorbitol,sucrose, mannitol, trehalose, or xylitol in an amount that facilitatesdispersal of the powder from the device, typically from about 1 wt. % orless to 99 wt. % or more of the formulation, preferably from about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75,80, 85, or 90 wt. % of the formulation.

In some embodiments, the invention is directed to kits comprising one ormore vaccine compositions of the invention. Such kits can be provided toan administering physician or other health care professional.

In some embodiments, the kit is a package that houses one or morecontainers which comprises one or more vaccine compositions andinstructions for administering the vaccine composition to a subject. Insome embodiments, the kit can also comprise one or more othertherapeutic agents. The kit can optionally contain one or morediagnostic tools and instructions for use.

In some embodiments, the kit comprises an immunization schedule. In someembodiments, a vaccine cocktail containing two or more vaccinecompositions can be included, or separate pharmaceutical compositionscontaining different vaccines or therapeutic agents. The kit can alsocontain separate doses of the vaccine composition for serial orsequential administration.

In some embodiments, the kit further comprises suitable deliverydevices, e.g., syringes, inhalation devices, and the like, along withinstructions for administrating the therapeutic agents. The kit canoptionally contain instructions for storage, reconstitution (ifapplicable), and administration of any or all therapeutic agentsincluded. The kits can include a plurality of containers reflecting thenumber of administrations to be given to a subject. If the kit containsa first and second container, then a plurality of these can be present.

Methods of Treatment

Another aspect of the invention is directed to a method of inducing animmune response, comprising administering to a subject in need thereofan immunologically-effective amount of the above-mentioned conjugate orcomposition. In some embodiments, the surface polysaccharide antigen isan O polysaccharide (OPS), a core oligosaccharide and an Opolysaccharide (COPS), a capsule polysaccharide, or combinationsthereof. In some embodiments of the invention, the surfacepolysaccharide antigen is an O polysaccharide antigen (OPS). The surfacepolysaccharide antigen and the flagellin can be covalently linked.

In some embodiments, methods of the claimed invention includeadministering multiple conjugates comprising one or more Pseudomonasflagellins or antigenic fragments or derivatives thereof covalentlylinked to one or more Klebsiella O polysaccharides (OPS) to induce animmune response. The multiple conjugates can comprise two differentPseudomonas flagellin covalently linked to one or more Klebsiella Opolysaccharides (OPS). The two different Pseudomonas flagellins can be aPseudomas aeruginosa flagellin type A (FlaA) and a Pseudomonasaeruginosa flagellin type B (FlaB).

In further embodiments of the method, the multiple conjugates cancomprise four different OPS antigens from Klebsiella pneumoniae. Forexample, the four different OPS can be derived from Klebsiellapneumoniae serovars O1, O2a, O3 and O5. Further, the two differentPseudomonas flagellins can be Pseudomonas aeruginosa flagellin type A(FlaA) and Pseudomas aeruginosa flagellin type B (FlaB) and/or the fourKlebsiella OPS can be from Klebsiella pneumoniae serovars O1, O2a, O3and O5. The Pseudomonas flagellin can be covalently linked to one ormore OPS from a single Klebsiella pneumoniae serovar type or can becovalently linked to OPS from multiple Klebsiella pneumoniae serovartypes. The Pseudomonas aeruginosa flagellin type A (FlaA) can compriseSEQ ID NO:1 and/or the Pseudomas aeruginosa flagellin type B (FlaB) cancomprise SEQ ID NO:2.

In some embodiments, the conjugate or composition is administeredmultiple times to the subject. The conjugate or composition may also beadministered a single time to the subject. The term “subject” as usedherein, refers to animals, such as mammals. For example, mammalscontemplated include humans, primates, dogs, cats, sheep, cattle, goats,pigs, horses, chickens, mice, rats, rabbits, guinea pigs, and the like.The terms “subject”, “patient”, and “host” are used interchangeably.

Human subjects are not limiting and can include deployed soldiers,hospital workers, patients and residents of chronic care facilities. Insome embodiments, the patient is in a hospital or in a skilled nursingfacility. In some embodiments, the subject is administered the conjugateor composition prior to, during, or after a surgery. The surgery is notlimiting and can be, for example, colon surgery, hip arthroplasty, orsmall-bowel surgery. Further, the conjugate or composition can beadministered prior to, during, or after a procedure selected fromcentral venous catheterization, urinary tract catheterization, andintubation with a ventilator tube.

As used herein, an “immune response” is the physiological response ofthe subject's immune system to an immunizing composition. An immuneresponse may include an innate immune response, an adaptive immuneresponse, or both. In some embodiments of the present invention, theimmune response is a protective immune response. A protective immuneresponse confers immunological cellular memory upon the subject, withthe effect that a secondary exposure to the same or a similar antigen ischaracterized by one or more of the following characteristics: shorterlag phase than the lag phase resulting from exposure to the selectedantigen in the absence of prior exposure to the immunizing composition;production of antibody which continues for a longer period thanproduction of antibody resulting from exposure to the selected antigenin the absence of prior exposure to the immunizing composition; a changein the type and quality of antibody produced in comparison to the typeand quality of antibody produced upon exposure to the selected antigenin the absence of prior exposure to the immunizing composition; a shiftin class response, with IgG antibodies appearing in higherconcentrations and with greater persistence than IgM, than occurs inresponse to exposure to the selected antigen in the absence of priorexposure to the immunizing composition; an increased average affinity(binding constant) of the antibodies for the antigen in comparison withthe average affinity of antibodies for the antigen resulting fromexposure to the selected antigen in the absence of prior exposure to theimmunizing composition; and/or other characteristics known in the art tocharacterize a secondary immune response.

In some embodiments, the immunogenicity of the conjugates andcompositions of the invention are greater than the immunogenicity of atleast one of the surface polysaccharide antigen or flagellin protein oran antigenic fragment or a derivative thereof alone. Methods ofmeasuring immunogenicity are well known to those in the art andprimarily include measurement of serum antibody including measurement ofamount, avidity, and isotype distribution at various times afterinjection of the conjugate vaccine. Greater immunogenicity may bereflected by a higher titer and/or increased life span of the antibodiesImmunogenicity may also be measured by the ability to induce protectionto challenge with noxious substances or virulent organismsImmunogenicity may also be measured by the ability to immunize neonataland/or immune deficient mice Immunogenicity may be measured in thepatient population to be treated or in a population that mimics theimmune response of the patient population.

In some embodiments, the immune response that is generated by theconjugates and compositions of the invention is a protective immuneresponse against infection by one or more Klebsiella and/or Pseudomonasserovars, including those serovars described herein.

In some embodiments, the conjugates and compositions of the inventionare administered alone in a single dose or administered in sequentialdoses. In other aspects of the invention, the conjugates andcompositions of the invention are administered as a component of ahomologous or heterologous prime/boost regimen in conjunction with oneor more vaccines. In some embodiments of the invention, a single boostis used. In some embodiments of the invention, multiple boostimmunizations are performed. In particular aspects of the inventiondrawn to a heterologous prime/boost, a mucosal bacterialprime/parenteral conjugate boost immunization strategy is used. In someembodiments, one or more (or all) of the live (or killed) attenuatedSalmonella enterica serovars used as a reagent strain to expressPseudomonas flagellin as taught herein can be administered orally to asubject and the subject can be subsequently boosted parenterally with aconjugates and compositions of the invention as described herein. Insome embodiments, one or more (or all) of the live (or killed)attenuated Klebsiella used as a reagent strain to isolate surfacepolysaccharide as taught herein can be administered orally to a subjectand the subject can be subsequently boosted parenterally with aconjugates and compositions of the invention as described herein.

In practicing immunization protocols for treatment and/or prevention, animmunologically-effective amount of conjugates and compositions of theinvention are administered to a subject. As used herein, the term“immunologically-effective amount” means the total amount of therapeuticagent (e.g., conjugate or composition) or other active component that issufficient to show an enhanced immune response in the subject. When“immunologically-effective amount” is applied to an individualtherapeutic agent administered alone, the term refers to thattherapeutic agent alone. When applied to a combination, the term refersto combined amounts of the ingredients that result in the therapeuticeffect, whether administered in combination, serially or simultaneously,and regardless of order of administration.

The particular dosage depends upon the age, weight, sex and medicalcondition of the subject to be treated, as well as on the method ofadministration. Suitable doses can be readily determined by those ofskill in the art.

The conjugates and compositions of the invention can be administered byeither single or multiple dosages of an effective amount. In someembodiments, an effective amount of the compositions of the inventioncan vary from 0.01-5,000 μg/ml per dose. In other embodiments, aneffective amount of the conjugate or composition of the invention canvary from 0.1-500 μg/ml per dose, and in other embodiments, it can varyfrom 10-300 μg/ml per dose. In one embodiment, the dosage of theconjugate or composition administered will range from about 10μg toabout 1000 μg. In another embodiment, the amount administered will bebetween about 20 μg and about 500 μg. In some embodiments, the amountadministered will be between about 75 μg and 250 μg. Greater doses maybe administered on the basis of body weight. The exact dosage can bedetermined by routine dose/response protocols known to one of ordinaryskill in the art.

In some embodiments, the amount of conjugates and compositions of theinvention that provide an immunologically-effective amount forvaccination against Klebsiella and/or Pseudomonas infections is fromabout 1 μg or less to about 100 μg or more. In some embodiments, it isfrom about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50μg to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 μg per kg body weight.In some embodiments, the immunologically-effective amount forvaccination against Klebsiella and/or Pseudomonas infection is from 0.01μg to 10 μg.

The conjugates and compositions of the invention may confer resistanceto Klebsiella and/or Pseudomonas infections by either passiveimmunization or active immunization. In one embodiment of passiveimmunization, the conjugate or composition is provided to a subject(i.e. a human or mammal), and the elicited antisera is recovered anddirectly provided to a recipient suspected of having an infection causedby Klebsiella and/or Pseudomonas.

In some embodiments, the present invention provides a means forpreventing or attenuating infection by Klebsiella and/or Pseudomonas orby organisms which have antigens that can be recognized and bound byantisera to the polysaccharide and/or protein of the conjugate orcomposition.

The administration of the conjugate or composition (or the antiserawhich it elicits) may be for either a “prophylactic” or “therapeutic”purpose. When provided prophylactically, the conjugate or composition isprovided in advance of any symptom of Klebsiella and/or Pseudomonasinfection. The prophylactic administration of the conjugate orcomposition serves to prevent or attenuate any subsequent infection.When provided therapeutically, the conjugate or composition is providedupon the detection of a symptom of actual infection. The therapeuticadministration of the conjugate or composition serves to attenuate anyactual infection.

The conjugate or composition of the invention may, thus, be providedeither prior to the onset of infection (so as to prevent or attenuate ananticipated infection) or after the initiation of an actual infection.

The conjugate or composition of the invention may be administered towarm-blooded mammals of any age. The conjugate or composition can beadministered as a single dose or in a series including one or moreboosters. In some embodiments, the immunization schedule would involve aprimary series of three immunizations with a spacing of 1-2 monthsbetween the doses. In some settings a booster dose could be administered˜6-12 months later. For example, an infant can receive three doses at 6,10 and 14 weeks of age (schedule for infants in sub-Saharan Africa) orat 2, 4, and 6 months of life (schedule for U.S. infants). In someembodiments, U.S. infants might receive a booster at 12-18 months ofage. Another target population would be U.S. elderly who would likelyreceive 2-3 doses spaced 1-2 months apart. A further target populationwould be patients upon admission to a hospital.

Methods of Making the Conjugate

The methods that can be used to make the conjugates of the invention arenot limiting. Methods useful for producing conjugate vaccines have beenpreviously described and disclosed in U.S. Pat. No. 4,673,574, U.S. Pat.No. 4,789,735, U.S. Pat. No. 4,619,828, U.S. Pat. No. 4,284,537, U.S.Pat. No. 5,370,872, U.S. Pat. No. 5,302,386, U.S. Pat. No. 5,576,002,and U.S. Patent Application Pub. No. 2011/0274714, all of whichdisclosures are incorporated herein by reference.

In one embodiment, the invention is directed towards a method of makingthe conjugates described herein comprising binding a Klebsiella surfacepolysaccharide antigen and a Pseudomonas flagellin protein or anantigenic fragment or a derivative thereof. In some embodiments, thebinding is covalent. In some embodiments, the surface polysaccharideantigen is an O polysaccharide (OPS). Further embodiments includecovalently bonding Pseudomas aeruginosa flagellin type A (FlaA) and/orPseudomas aeruginosa flagellin type B (FlaB) to at least one OPS fromKlebsiella pneumoniae serovars O1, O2a, O3 and O5 to arrive at theconjugates described herein.

In some embodiments, the surface polysaccharide antigen is isolated froma Klebsiella pneumoniae serovar having one or more mutations. Forexample, the Klebsiella pneumoniae may have an attenuating mutation inthe guaBA locus and/or a mutation in the wza-wzc locus.

In some embodiments, the Pseudomonas flagellin protein is isolated froma heterologous Gram-negative bacteria (GNB) expression system, includingSalmonella and Escherichia coli. In some embodiments, the flagellinprotein is isolated from a Salmonella enterica serovar strain engineeredto express Pseudomonas aeruginosa flagellin protein. In someembodiments, the Salmonella enterica serovar is Enteritidis. In someembodiments, the Salmonella enterica serovar strain may have anattenuating mutation, for example, in the guaBA locus. In someembodiments, the flagellin is purified from the bacterial supernatant ofthe Salmonella enterica serovar reagent strains described herein bychromatographic methods.

The Pseudomas aeruginosa flagellin can be purified and isolated usingconventional techniques and methods. Such methods can include mechanicalshearing, removal at low pH, heating or purification from bacterialsupernatants. Methods of purification of a flagellin protein from wholeflagella are known in the art or can be readily modified by one ofordinary skill in the art using methods known in the art. For example,by modifying the method of Ibrahim et al., purification of flagella isachieved; below pH 3.0, flagella dissociate into flagellin subunits(Ibrahim et al. J. Clin. Microbiol. 1985; 22:1040-4). Further methodsfor purification include adaptation of the mechanical shearing, andsequential centrifugation steps for purification of flagellin inflagella from bacterial cells.

In some embodiments, COPS and OPS can be isolated by methods including,but not limited to mild acid hydrolysis removal of lipid A from LPS.Other embodiments may include use of hydrazine as an agent for COPS orOPS preparation. Preparation of LPS can be accomplished by known methodsin the art. In some embodiments, LPS is prepared according to methods ofDarveau et al. J. Bacteriol., 155(2):831-838 (1983), or Westphal et al.Methods in Carbohydrate Chemistry. 5:83-91 (1965) which are incorporatedby reference herein.

In some embodiments, the LPS is purified by a modification of themethods of Darveau et al., supra, followed by mild acid hydrolysis.

The surface polysaccharide antigen and flagellin can be conjugated usingknown techniques and methods. For example, techniques to conjugatesurface polysaccharide antigen and flagellin can include, in part,coupling through available functional groups (such as amino, carboxyl,thiol and aldehyde groups). See, e.g., Hermanson, BioconjugateTechniques (Academic Press; 1992); Aslam and Dent, eds. Bioconjugation:Protein coupling Techniques for the Biomedical Sciences (MacMillan:1998); S. S. Wong, Chemistry of Protein Conjugate and Crosslinking CRCPress (1991), and Brenkeley et al., Brief Survey of Methods forPreparing Protein Conjugates With Dyes, Haptens and Cross-LinkingAgents, Bioconjugate Chemistry 3 #1 (Jan. 1992).

In some embodiments of the present invention, the surface polysaccharideantigen and flagellin or fragments or derivatives thereof, can includefunctional groups or, alternatively, can be chemically manipulated tobear functional groups. In some embodiments, the presence of functionalgroups can facilitate covalent conjugation. Such functional groups caninclude amino groups, carboxyl groups, aldehydes, hydrazides, epoxides,and thiols, for example. Functional amino and sulfhydryl groups can beincorporated therein by conventional chemistry. Primary amino groups canbe incorporated by reaction with ethylenediamine in the presence ofsodium cyanoborohydride and sulfhydryls may be introduced by reaction ofcysteamine dihydrochloride followed by reduction with a standarddisulfide reducing agent.

Flagellin may contain amino acid side chains such as amino, carbonyl,hydroxyl, or sulfhydryl groups or aromatic rings that can serve as sitesfor conjugation. Residues that have such functional groups can be addedto either the surface polysaccharide antigen or flagellin. Such residuesmay be incorporated by solid phase synthesis techniques or recombinanttechniques, for example.

Surface polysaccharide antigen and flagellin can be chemicallyconjugated using conventional crosslinking agents such as carbodiimides.Examples of carbodiimides are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide (CMC), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC),and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

Examples of other crosslinking agents are cyanogen bromide,glutaraldehyde and succinic anhydride. In general, any of a number ofhomobifunctional agents including a homobifunctional aldehyde, ahomobifunctional epoxide, a homobifunctional imidoester, ahomobifunctional N-hydroxysuccinimide ester, a homobifunctionalmaleimide, a homobifunctional alkyl halide, a homobifunctional pyridyldisulfide, a homobifunctional aryl halide, a homobifunctional hydrazide,a homobifunctional diazonium derivative or a homobifunctionalphotoreactive compound can be used. Also included are heterobifunctionalcompounds, for example, compounds having an amine-reactive and asulfhydryl-reactive group, compounds with an amine-reactive and aphotoreactive group, and compounds with a carbonyl-reactive and asulfhydryl-reactive group.

Specific examples of homobifunctional crosslinking agents include thebifunctional N-hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyltartarate; the bifunctional imidoesters dimethyl adipimidate, dimethylpimelimidate, and dimethyl suberimidate; the bifunctionalsulfhydryl-reactive crosslinkers 1,4-di-[3′-(2′-pyridyldithio)propion-amidolbutane, bismaleimidohexane, andbis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamide)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adiphaldehyde; a bifunctional epoxied such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

Examples of other common heterobifunctional crosslinking agents that maybe used include, but are not limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodacteyl) aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(-maleimidobutyryloxy)succinimide ester), MPHB(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio) propionate). For example,crosslinking may be accomplished by coupling a carbonyl group to anamine group or to a hydrazide group by reductive amination.

In another aspect of the invention, the surface polysaccharide antigenand flagellin can be conjugated through polymers, such as PEG,poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, immunoglobulins,and copolymers of D-lysine and D-glutamic acid. Conjugation of thesurface polysaccharide antigen and flagellin may be achieved in anynumber of ways, including involving one or more crosslinking agents andfunctional groups on the surface polysaccharide antigen and/orflagellin. The polymer can be derivatized to contain functional groupsif it does not already possess appropriate functional groups.

In some embodiments, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP) conjugation chemistry is used to achieve efficient synthesis ofthe surface polysaccharide antigen and flagellin conjugates. In someembodiments, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)is used to conjugate OPS-FlaA conjugates and OPS-FlaB conjugates.

In some embodiments, the surface polysaccharide antigen or flagellin isconjugated to a linker prior to conjugation. In some embodiments, thelinker is adipic acid dihydrazide (ADH). The present inventioncontemplates the use of any linker capable of conjugating the surfacepolysaccharide antigen to flagellin. In some embodiments, the presenceof a linker promotes optimum immunogenicity of the conjugate andcomposition and more efficient coupling. In some embodiments, thelinkers separate the two or more antigenic components by chains whoselength and flexibility can be adjusted as desired. Between thebifunctional sites, the chains can contain a variety of structuralfeatures, including heteroatoms and cleavage sites. In some embodiments,linkers also permit corresponding increases in translational androtational characteristics of the antigens, increasing access of thebinding sites to soluble antibodies. Besides ADH, suitable linkersinclude, for example, heterodifunctional linkers such as e-aminohexanoicacid, chlorohexanol dimethyl acetal, D-glucuronolactone andp-nitrophenyl amine. Coupling reagents contemplated for use in thepresent invention include hydroxysuccinimides and carbodiimides. Manyother linkers and coupling reagents known to those of ordinary skill inthe art are also suitable for use in the invention. Such compounds arediscussed in detail by Dick et al., Conjugate Vaccines, J. M. Cruse andR. E. Lewis, Jr., eds., Karger, N.Y., pp. 48-114, hereby incorporated byreference.

In some embodiments, ADH is used as the linker. In some embodiments, themolar ratio of ADH to surface polysaccharide antigen such as OPS in thereaction mixture is typically between about 10:1 and about 250:1. Insome embodiments, a molar excess of ADH is used to ensure more efficientcoupling and to limit OPS-OPS coupling. In some embodiments, the molarratio is between about 50:1 and about 150:1. In other embodiments, themolar ratio is about 100:1. Similar ratios of AH-OPS to the flagellin inthe reaction mixture are also contemplated. In some embodiments, one ADHper OPS is present in the AH-OPS conjugate.

Other linkers are available and can be used to link two aldehydemoieties, two carboxylic acid moieties, or mixtures thereof. Suchlinkers include (C₁-C₆) alkylene dihydrazides, (C₁-C₆) alkylene orarylene diamines, -aminoalkanoic acids, alkylene diols or oxyalkenediols or dithiols, cyclic amides and anhydrides and the like. Forexamples, see U.S. Pat. No. 5,739,313, incorporated herein in itsentirety.

In some embodiments, conjugation is conducted at a temperature of fromabout 0° C. to about 5° C. for about 36 to about 48 hours. In oneembodiment, conjugation is conducted at about 4° C. for about 36 hours,followed by about an additional 18 to 24 hours at a temperature of fromabout 20° C. to about 25° C. In another embodiment, conjugation isconducted for about 18 hours at about 20 to 24° C., such that theresidual cyanate groups react with water and decompose. Longer orshorter conjugation times and/or higher or lower conjugationtemperatures can be employed, as desired. In some embodiments, it isdesirable, however, to conduct the conjugation reaction, at leastinitially, at low temperatures, for example, from about 0° C. to about5° C., such as about 4° C., so as to reduce the degree of precipitationof the conjugate.

In some embodiments of the invention, conjugation of the surfacepolysaccharide antigen and flagellin protein is on the terminal aminogroup of lysine residues. In some embodiments of the invention,conjugation is to cysteine groups. In some embodiments of the invention,conjugation of the surface polysaccharide antigen is to N-terminalserine groups. In some embodiments of the invention, conjugation of thesurface polysaccharide antigen to the flagellin is directed towards theC-terminal carboxylic acid group. In some embodiments of the invention,conjugation is to naturally occurring amino acid groups. In otherembodiments of the invention, conjugation is to engineered amino acidsequences and residues within the flagellin protein.

In some embodiments of the invention, conjugation of the surfacepolysaccharide antigen and flagellin is on random free hydroxyl groupson the OPS polysaccharide chain. In some embodiments of the invention,conjugation of the flagellin to the surface polysaccharide antigen andis at the terminal end of the polysaccharide chain.

In some embodiments of the invention, the surface polysaccharide antigenand flagellin reactants contain multiple reactive groups per molecule.In some embodiments, an activated surface polysaccharide antigenmolecule can react with and form more than one linkage to more than oneflagellin. Likewise, an activated flagellin can react with and form morethan one linkage to more than one activated surface polysaccharideantigen. Therefore, in some embodiments, the conjugate product is amixture of various cross-linked matrix-type lattice structures. Forexample, a single linkage can be present, or 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 or more linkages can be present. The average number oflinkages between a surface polysaccharide and flagellin antigen can beadjusted, as desired. In some embodiments, the average number oflinkages can depend upon the type of OPS polysaccharide, the type offlagellin protein, the conjugation method, the reaction conditions, andthe like.

In some embodiments, purification processes such as columnchromatography and/or ammonium sulfate precipitation of the conjugatefrom unconjugated polysaccharide may not be necessary. However, incertain embodiments it can be desirable to conduct one or morepurification steps. In some embodiments, after conjugation, theconjugate can be purified by any suitable method. Purification can beemployed to remove unreacted polysaccharide, protein, or small moleculereaction byproducts. Purification methods include ultrafiltration, sizeexclusion chromatography, density gradient centrifugation, hydrophobicinteraction chromatography, ammonium sulfate fractionation, ion exchangechromatography, ligand exchange chromatography, immuno-affinitychromatography, polymyxin-b chromatography, and the like, as are knownin the art. In some embodiments, the conjugation reactions proceed withhigher yield, and generate fewer undesirable small molecule reactionbyproducts. Accordingly, in some embodiments no purification may benecessary, or only a minor degree of purification can be desirable. Theconjugate or composition of the invention can be concentrated ordiluted, or processed into any suitable form for use in pharmaceuticalcompositions, as desired.

Genetically Engineered Strains

In another embodiment, the invention provides a modified Klebsiella thatis useful for isolating the surface polysaccharide antigen for use inmaking the conjugates of the invention. In some embodiments, themodified Klebsiella is a modified Klebsiella pneumonia. In someembodiments, the modified Klebsiella comprises one or more attenuatingmutations. In some embodiments, the modified Klebsiella has anattenuating mutation in the guaBA locus. In some embodiments, theKlebsiella comprises one or more mutations in the wza-wzc locus. In someembodiments, the Klebsiella pneumoniae serovar can be O1, O2a, O3,and/or O5. In some embodiments, the Klebsiella is Klebsiella pneumoniaeserovar O1, O2a, O3, or O5 having an attenuating mutation in the guaBAlocus and a mutation in the wza-wzc locus.

In some embodiments the guaA gene (NCBI-ProteinID: ABR78243 NCBI-GI:152971364 NCBI-GeneID: 5339904 UniProt: A6TCC2) of Klebsiella pneumoniaecomprises SEQ ID NO:5, and encodes guanosine monophosphate synthase.

In some embodiments the guaB gene (NCBI-ProteinlD: ABR78244 NCBI-GI:152971365 NCBI-GeneID: 5339905 UniProt: A6TCC3) of Klebsiella pneumoniaecomprises SEQ ID NO:6, and encodes inosine 5′-monophosphatedehydrogenase.

In some embodiments the wza gene (NCBI-ProteinID: ABR77930 NCBI-GI:152971051 NCBI-GeneID: 5340218 UniProt: A6TBF9) of Klebsiella pneumoniaecomprises SEQ ID NO:7, and encodes capsule export-outer membraneprotein.

In some embodiments the wzb gene (NCBI-ProteinID: ABR77929 NCBI-GI:152971050 NCBI-GeneID: 5340217 UniProt: A6TBF8) of Klebsiella pneumoniaecomprises SEQ ID NO:8, and encodes protein tyrosine phosphatase.

In some embodiments the K2-wzc gene (NCBI-ProteinID: ABR77928 NCBI-GI:152971049 NCBI-GeneID: 5340932 UniProt: A6TBF7) of Klebsiella pneumoniaecomprises SEQ ID NO:9, and encodes tyrosine autokinase.

In another embodiment, the invention provides a modified Gram-negativebacteria (GNB) engineered to express Pseudomonas flagellin which can beisolated for use in preparation of the conjugates of the invention. Insome embodiments, the Gram-negative bacteria is Escherichia coli. Insome embodiments, the Gram-negative bacteria is a Salmonella such as aSalmonella enterica serovar strain. In some embodiments, the Salmonellaenterica serovar is selected from Enteritidis, Typhimurium, andParatyphi A. In some embodiments, the Salmonella enterica serovar isEnteritidis.

In some embodiments, the Gram-negative bacteria expressing Pseudomonasflagellin has one or more mutations. In some embodiments, theGram-negative bacteria has one or more mutations in the guaBA locus, theguaB gene, the guaA gene, the clpP gene, the clpX gene and/or the clpPXlocus. In some embodiments, the Gram-negative bacteria expressingPseudomonas flagellin has one or more codon optimized Pseudomonas fliCgenes. In some embodiments, the Gram-negative bacteria expressingPseudomonas flagellin encodes a excretion signal for flagellin.

In some embodiments, the Gram-negative bacteria, such as Salmonellaenterica, has at least one attenuating mutation in the guaBA locusand/or the clpPX locus. In some embodiments, one or more of guaBA, clpPXand fliD are mutated to create highly attenuated strains thathyper-secrete flagellin monomers into the supernatant. A guaBA mutation(involved in guanosine nucleotide synthesis (Samant S et al., PLoSPathog. 2008; 4(2):e37)) is highly attenuating in several Gram negativepathogens (e.g., Shigella (Kotloff K L et al., Hum Vaccin. 2007;3(6):268-275), Salmonella (Tennant S M et al., Infect Immun. 2011;79(10):4175-4185; Gat O et al., PLoS Negl Trop Dis. 2011; 5(11):e1373),Francisella (Santiago A E et al., Vaccine. 2009; 27(18):2426-2436)).When either clpP or clpX (that form the ClpPX protease) is deleted, themaster flagella regulator complex FlhD/F1hC is not degraded and largeamounts of flagella are produced. Deletion of clpPX is alsoindependently attenuating (Tennant S M et al., Infect Immun. 2011;79(10):4175-4185; Tomoyasu T et al., J Bacteriol. 2002; 184(3):645-653).Deletion of the gene for the flagella capping protein FliD causesflagellin monomers to be exported into the supernatant, and engineeredSalmonella mutants deficient in clpPX and fliD produce and export largeamounts of flagellin into the culture supernatant. These recombinantstrains are considered as safe from an occupational health and safetyperspective and enable conjugate vaccine carrier proteins to beexpressed at high levels, thus lowering the overall cost of manufacture.

Growth conditions in fully chemically defined minimal media forattenuated S. Enteritidis and S. Typhimurium strains have beenestablished, whereby an optical density at 600 nm (OD₆₀₀) of 15-18 isconsistently attained at 20 L fermentation scale. Prototype attenuatedS. Enteritidis reagent strain CVD 1943 ΔguaBA ΔclpP ΔfliD wasconstructed from wild-type strain S. Enteritidis R11 (a Malian clinicalisolate) (Richmond P, J Infect Dis. 2000; 181(2):761-764).

In some embodiments, the Gram negative bacteria has an inactivatingmutation in fliC such as a deletion in fliC. Such strain may furtherhave an inserted (either in the chromosome or on a plasmid) heterologousfliC such as fliC from Pseudomas aeruginosa or a bacteria producingflagellin with cross-reactivity to fliC from Pseudomonas aeruginosa.

In some embodiments, the Gram negative bacteria is Salmonella entericahaving a mutation in fliC and having a plasmid encoding Pseudomonasaeruginosa Type A flagellin (F1aA) and/or Pseudomas aeruginosa Type Bflagellin (FlaB). In some embodiments, the amino acid sequence of FlaAcomprises SEQ ID NO:1 and the nucleotide sequence of FlaA comprises SEQID NO:3. In some embodiments, the amino acid sequence of FlaB comprisesSEQ ID NO:2 and the nucleotide sequence comprises SEQ ID NO:4. In someembodiments, the Salmonella enterica expressing Pseudomonas flagellinhas one or more codon optimized Pseudomonas fliC genes. In someembodiments, the Salmonella enterica expressing Pseudomonas flagellinencodes a Salmonella enterica Enteritidis fliC excretion signal.

In some embodiments, the Gram negative bacteria hyper-secretesPseudomonas flagellin. In some embodiments, the Gram negative bacteriacomprises a clpP or clpX (that form the ClpPX protease) mutation causingthe master flagella regulator complex FlhD/FlhC to not be degraded,thereby causing the production of large amounts of flagella.

In some embodiments, using modified strains with attenuating mutationscan simplify purification. Attenuated Salmonella strains are consideredas safe from an occupational health and safety perspective. As usedherein, attenuated strains are those that have a reduced, decreased, orsuppressed ability to cause disease in a subject, or those completelylacking in the ability to cause disease in a subject. Attenuated strainsmay exhibit reduced or no expression of one or more genes, may expressone or more proteins with reduced or no activity, may exhibit a reducedability to grow and divide, or a combination of two or more of thesecharacteristics.

In some embodiments, the attenuated strains producing Pseudomonasflagellin of the invention have a mutation in one or more of the guaBAlocus, the guaB gene, the guaA gene, the clpP gene, the clpX gene andthe clpPX locus. For example, the attenuated strains can have a mutation(i) in the guaB gene and the clpP gene, (ii) in the guaA gene and theclpP gene, (iii) in the guaBA locus, and the clpP gene, (iv) in the guaBgene and the clpX gene, (v) in the guaA gene and the clpX gene, (vi) inthe guaBA locus, and the clpX gene, (vii) in the guaB gene and the clpPXlocus, (viii) in the guaA gene and the clpPX locus, or (ix) in both theguaBA locus and the clpPX locus.

In some embodiments, attenuated strains are prepared having inactivatingmutations (such as chromosomal deletions) in both the guaBA locus(encoding enzymes involved in guanine nucleotide biosynthesis) and theclpPX locus (encoding an important metabolic ATPase) genes. In someembodiments, one or more of the attenuated strains also have fliD andfliC mutations.

The mutations of the loci and genes described herein can be anymutation, such as one or more nucleic acid deletions, insertions orsubstitutions. The mutations can be any deletion, insertion orsubstitution of the loci or genes that results in a reduction or absenceof expression from the loci or genes, or a reduction or absence ofactivity of a polypeptide encoded by the loci or genes. The mutationsmay be in the coding or non-coding regions of the loci or genes.

In some embodiments, the chromosomal genome of the Gram negativebacteria or Klebsiella is modified by removing or otherwise modifyingthe guaBA locus, and thus blocking the de novo biosynthesis of guaninenucleotides. In some embodiments, a mutation in the guaBA locusinactivates the purine metabolic pathway enzymes IMP dehydrogenase(encoded by guaB) and GMP synthetase (encoded by guaA). In someembodiments, the strains are unable to de novo synthesize GMP, andconsequently GDP and GTP nucleotides, which severely limits bacterialgrowth in mammalian tissues. The ΔguaBA mutants of the present inventionare unable to grow in minimal medium unless supplemented with guanine.

In some embodiments, the guaA gene of S. Enteritidis, which encodes GMPsynthetase, is 1578 bp in size (GenBank Accession Number NC_011294.1(2623838-2625415) (SEQ ID NO:10). In some embodiments, the guaA gene ofS. Typhimurium, is 1578 bp in size (GenBank Accession Number NC_003197.1(2622805..2624382, complement) (SEQ ID NO:11). In some embodiments, theguaA gene of S. Typhi, is 1578 bp in size (GenBank Accession NumberNC_004631.1 (415601.417178) (SEQ ID NO:12). In some embodiments, theguaA gene of S. Paratyphi A, is 1578 bp in size (GenBank AccessionNumber NC_006511.1 (421828..423405) (SEQ ID NO:13). In some embodiments,the guaA gene of S. Paratyphi B is 1578 bp in size (GenBank AccessionNumber NC_010102.1 (418694.420271) (SEQ ID NO:14).

Deletion mutants can be produced by eliminating portions of the codingregion of the guaA gene so that proper folding or activity of GuaA isprevented. For example, about 25 to about 1500 bp, about 75 to about1400 bp, about 100 to about 1300 bp, or all of the coding region can bedeleted. Alternatively, the deletion mutants can be produced byeliminating, for example, a 1 to 100 bp fragment of the guaA gene sothat the proper reading frame of the gene is shifted. In the latterinstance, a nonsense polypeptide may be produced or polypeptidesynthesis may be aborted due to a frame-shift-induced stop codon. Thepreferred size of the deletion removes both guaB and guaA, from the ATGstart codon of guaB to the stop codon of guaA.

In some embodiments, the guaB gene of S. Enteritidis which encodes IMPdehydrogenase, is 1467 bp in size (GenBank Accession NumberNC_(—O)11294.1 (2625485-2626951, complement) (SEQ ID NO:15). In someembodiments, the guaB gene of S. Typhimurium is 1467 bp in size (GenBankAccession Number NC_003197.1 (2624452.2625918, complement) (SEQ IDNO:16). In some embodiments, the guaB gene of S. Paratyphi A is 1467 bpin size (GenBank Accession Number NC_006511.1 (420292.421758) (SEQ IDNO:17). Deletion mutants can be produced by eliminating portions of thecoding region of the guaB gene so that proper folding or activity ofGuaB is prevented. For example, about 25 to about 1400 bp, about 75 toabout 1300 bp, about 100 to about 1200 bp, or all of the coding regioncan be deleted. Alternatively, the deletion mutants can be produced byeliminating, for example, a 1 to 100 bp fragment of the guaB gene sothat the proper reading frame of the gene is shifted. In the latterinstance, a nonsense polypeptide may be produced or polypeptidesynthesis may be aborted due to a frame-shift-induced stop codon. Thepreferred size of the deletion removes both guaB and guaA, from the ATGstart codon of guaB to the stop codon of guaA.

In some embodiments, the clpP gene of S. Enteritidis, which encodes aserine-protease, is 624 bp in size (GenBank Accession Number NC_011294.1(482580-483203) (SEQ ID NO:18). In some embodiments, the clpP gene of S.Typhimurium is 624 bp in size (GenBank Accession Number NC_003197.1(503210.503833) (SEQ ID NO:19). In some embodiments, the clpP gene of S.Paratyphi A is 624 bp in size (GenBank Accession Number NC_006511.1(2369275.2369898, complement) (SEQ ID NO:20).

Deletion mutants can be produced by eliminating portions of the codingregion of the clpP gene so that proper folding or activity of ClpP isprevented. For example, about 25 to about 600 bp, about 75 to about 500bp, about 100 to about 400 bp, or all of the coding region can bedeleted. Alternatively, the deletion mutants can be produced byeliminating, for example, a 1 to 100 bp fragment of the clpP gene sothat the proper reading frame of the gene is shifted. In the latterinstance, a nonsense polypeptide may be produced or polypeptidesynthesis may be aborted due to a frame-shift-induced stop codon. clpPforms an operon with clpX; the preferred size of the deletionencompasses only the downstream clpX gene and extends from the ATG startcodon to the stop codon, inclusive.

In some embodiments, the clpX gene of S. Enteritidis, which encodes achaperone ATPase, is 1272 bp in size (GenBank Accession NumberNC_011294.1 (483455-484726) (SEQ ID NO:21). In some embodiments, theclpX gene of S. Typhimurium is 1272 bp in size (GenBank Accession NumberNC_003197.1 (504085.505356) (SEQ ID NO:22). In some embodiments, theclpX gene of S. Paratyphi A is 1272 bp in size (GenBank Accession NumberNC_006511.1 (2367752.2369023, complement) (SEQ ID NO:23).

Deletion mutants can be produced by eliminating portions of the codingregion of the clpX gene so that proper folding or activity of ClpX isprevented. For example, about 25 to about 1200 bp, about 75 to about1100 bp, about 100 to about 1000 bp, or all of the coding region can bedeleted. Alternatively, the deletion mutants can be produced byeliminating, for example, a 1 to 100 bp fragment of the clpX gene sothat the proper reading frame of the gene is shifted. In the latterinstance, a nonsense polypeptide may be produced or polypeptidesynthesis may be aborted due to a frame-shift-induced stop codon. clpPforms an operon with clpX; the preferred size of the deletionencompasses only the downstream clpX gene and extends from the ATG startcodon to the stop codon, inclusive.

The fliC gene can be mutated using conventional techniques known in theart. The fliC gene encodes a flagellin protein. In some embodiments, thefliC gene from S. Enteritidis is 1518 bp in size (GenBank AccessionNumber NC_011294.1 (1146600.1148117) (SEQ ID NO:24). In someembodiments, the fliC gene of S. Typhimurium is 1488 bp in size (GenBankAccession Number NC_003197.1 (2047658.2049145, complement) (SEQ IDNO:25). In some embodiments, the fliC gene of S. Paratyphi A, is 1488 bpin size (GenBank Accession Number NC_006511.1 (989787.991274) (SEQ IDNO:26).

In some embodiments, deletions can be made in any of the loci or genesincluded herein by using convenient restriction sites located within theloci or genes, or by site-directed mutagenesis with oligonucleotides(Sambrook et al., Molecular Cloning, A Laboratory Manual, Eds., ColdSpring Harbor Publications (1989)).

In some embodiments, inactivation of the loci or genes can also becarried out by an insertion of foreign DNA using any convenientrestriction site, or by site-directed mutagenesis with oligonucleotides(Sambrook et al., supra) so as to interrupt the correct transcription ofthe loci or genes. The typical size of an insertion that can inactivatethe loci or genes is from 1 base pair to 100 kbp, although insertionssmaller than 100 kbp are preferable. In some embodiments, the insertioncan be made anywhere inside the loci or gene coding regions or betweenthe coding regions and the promoters. In some embodiments, the bacterialloci and genes are mutated using Lambda Red-mediated mutagenesis (see,e.g., Datsenko and Wanner, PNAS USA 97:6640-6645 (2000)).

While the invention has been described with reference to certainparticular examples and embodiments herein, those skilled in the artwill appreciate that various examples and embodiments can be combinedfor the purpose of complying with all relevant patent laws (e.g.,methods described in specific examples can be used to describeparticular aspects of the invention and its operation even though suchare not explicitly set forth in reference thereto).

EXAMPLES Example 1 Preparation and Testing of Conjugates ComprisingSurface Polysaccharides and Flagellin Proteins

Reagent strain to purify heterologous flagellins—We have created arecombinant reagent strain that can be used to purify large amounts ofheterologous flagellin by deleting fliC from the S. Enteritidis reagentstrain CVD 1943. The new reagent strain S. Enteritidis R11 AguaBA AclpPAfliD AfliC is designated CVD 1947. Heterologous fliC genes cansubsequently be cloned into pGEN206 (Stokes M G et al., Infection andImmunity, 2007; 75(4):1827-1834), a low copy number highly stableplasmid and introduced into CVD 1947.

Development of scalable upstream and downstream bioprocesses forobtaining purified flagellins and OPS—Robust, scalable, high yield andgeneralized purification methods have been developed to purify OPS andflagellins. We have developed and confirmed broadly applicable andscalable downstream manufacturing processes to purify secretedflagellins from culture supernatants, and OPS from bacterial cells usingcommon bioprocess methods and equipment. We have also confirmedperformance at 20 L scale for two different Salmonella serovar(Typhimurium and Enteritidis) where we can reliably purify to nearhomogeneity >150 mg of flagellin/L of supernatant, and ˜3 mg of COPS/gwet cell paste. By using fully chemically defined medium that does notcontain any exogenous biological material (e.g., peptides, proteins),all biological components originate from the bacterial strain, thusfurther simplifying flagellin purification. Notably, we have found thatsecreted flagellin represents the major (>90%) detectable proteinspecies in fermentation culture supernatant (FIG. 4). For flagellinpurification, protein can be purified by an initial capture directlyfrom fermentation supernatants onto cation exchange membranes. Asecondary anion exchange purification step, coupled with a finaltangential flow filtration step for buffer exchange and size selection,are sufficient to yield highly pure FliC (>500 mg/L from fermentationculture) with very low endotoxin levels (<0.1 EU/μg), and no detectableresidual nucleic acid. COPS extraction can be accomplished by a seriesof organic extraction steps coupled with ion exchange chromatography,TFT and ammonium sulfate precipitation steps, and purified to nearhomogeneity at a yield of ˜3 mg COPS/g wet cell paste (FIG. 4). We havesuccessfully used these bioprocess schemes to purify FliC flagellinsfrom Salmonella serovars Typhimurium, Enteritidis and Typhi, and COPSfrom S. Typhimurium and S. Enteritidis.

Development of methods to conjugate OPS with flagellin—We have developedseveral methods that can be used to simply and reliably conjugate OPSwith carrier proteins, and generate different types of conjugates.Salmonella COPS was successfully conjugated directly to the s-aminogroups of flagellin lysines or to carboxylic acid groups aftermodification with hydrazides, at random COPS hydroxyl groups along thepolysaccharide chain using 1-Cyano-4-dimethylaminopyridiniumtetrafluoroborate (CDAP), generating a lattice-type conjugate (FIG. 5,lane 4). End-linked sun-type conjugates have also been generated byconjugating at the carbonyl group present in the COPS ketocidic terminuswith amino-oxime thioether chemistry to Sulfo-GMBS(N-[γ-maleimidobutyryloxyl sulfosuccinimide ester) modified proteinlysines (FIG. 5, lane 5). Removal of unconjugated components andconjugation reagents can be accomplished by a 2-step purificationapproach developed at the Center for Vaccine Development (CVD),separating first by size with size-exclusion chromatography (SEC) andthen by charge using ion-exchange chromatography membranes. Theseconjugation methods have all been used successfully for the homologousCOPS and flagellins from S. Enteritidis and S. Typhimurium.

Immunogenicity of Salmonella COPS:Flagellin conjugates in mice-BALB/cmice immunized intramuscularly at days 0, 28 and 56 with 2.5 μg of S.Enteritidis COPS polysaccharide conjugated to S. Enteritidis FliCproduced significant levels of LPS and FliC specific serum IgG antibodytiters above the PBS controls Immunization with unconjugated COPS aloneor admixed with flagellin failed to produce anti-LPS IgG (FIG. 6).Importantly, mice immunized with COPS:FliC produced anti-FliC IgG titersthat were similar to those of mice immunized with COPS admixed withflagellin, suggesting that conjugation does not interfere withanti-flagellin immune responses. Post-vaccination COPS:FliC serarecognized LPS and FliC (FIG. 7), and sera from mice immunized withflagellin alone bound to Salmonella-associated flagella (FIG. 8). Wefurther determined that S. Enteritidis COPS:FliC conjugates synthesizedby either coupling at random polysaccharide hydroxyls via CDAP, orend-coupling at the terminal KDO carbonyl by aminooxy (oxime) chemistrygenerated similar levels of anti-LPS and anti-FliC serum IgG, andcomparably protected against fatal IP challenge with S. Enteritidis R11(TABLE 1).

TABLE 1 Immunogenicity of COPS:FliC conjugate vaccines in CD-1 mice andprotective efficacy against lethal challenge with wild-type S.Enteritidis R11^(a) Anti-FliC Anti-LPS GMT Anti-FliC GMT Anti-LPSMortality^(c) Vaccine Vaccine (EU^(d)/ml) Seropositive^(b) (EU/ml)Seropositive^(b) (dead/total) Efficacy PBS 101 0 82 0 12/13 — COPS:FliC8,735,020 100% 223 39%   3/13^(d) 75.0% CDAP COPS:FliC 9,548,869 100%392 40%   2/13^(d) 83.3% Oxime ^(a)IP LD₅ × 10⁵ CFU. ^(b)Defined as≧4-fold the GMT in mice immunized with PBS ^(c)Mice challenged i.p. with1 × 10⁶ CFU ^(d)p < 0.001 vs. PBS control animals by Fisher's exacttest. ^(d)ELISA Units

We also examined the effect of different COPS:FliC conjugate vaccinedoses on immunogenicity and efficacy (Chu C Y, Infect Immun. 1991;59(12):4450-4458). Maximal levels of anti-flagellin IgG and 100%seroconversion (≧4-fold vs. PBS geometric mean titers [GMT]) wereachieved at doses ≧0.25 μg of COPS:FliC. Mice immunized at the lowestdose of 0.025 μg COPS:FliC also displayed a significantly higher GMT ofanti-FliC IgG compared to mice receiving PBS, but with sub-maximallevels and several animals failing to produce detectable anti-FliC IgG(75% seropositive). We further observed that anti-flagellin IgGend-point titers were significantly higher than anti-LPS IgG levels inCOPS:FliC immunized mice at all doses tested. Immunization withCOPS:FliC doses of 10 μg and 2.5 μg elicited GMT's of 885 EU/ml and 308EU/ml respectively, whereas immunization with doses of 0.25 μg and 0.025μg resulted in GMT's of <80 EU/ml. Notably, whereas infection with 1×10⁶CFU of S. Enteritidis caused 100% mortality in the PBS control group,mice immunized with 0.025 μg, 0.25 μg, 2.5 μg or 10 μg of COPS:FliC wereall significantly protected (≧90% vaccine efficacy). We also found thatconjugation can reliably reduce TLRS stimulatory capacity, and that TLRSactivity was dispensable for immunogenicity. Our findings are inagreement with those reported for flagellin immunization experiments inTLRS-deficient mice, where anti-flagellin titers obtained werecomparable to wild-type mice (Sanders C J et al., Eur J Immunol. 2009;39(2):359-371). Vaxinnate Corporation has reported measurable rates ofadverse events at low dosage levels for their TLRS stimulatoryflagellin-based fusion proteins with influenza antigens (Turley C B etal., Vaccine. 2011; 29(32):5145-5152). Hence, we will aim to abolishTLR5 activity in our conjugates, as we have successfully done previously(Simon R, Infect Immun. 2011; 79(10):4240-4249).

Functional Activity of Vaccine Induced Antibodies

I. Opsonophagocytosis. Pooled sera from mice immunized with COPS:FliCwere able to cause uptake of wild-type and invA (invasion)-deficient S.Enteritidis R11 into J774 cultured mouse macrophage cells. Uptake wasreduced in the absence of bacterial expression of either flagellin(ΔfliC) or long-chain OPS (ΔrfaL) components present in the vaccine(FIG. 9) indicating that COPS:FliC vaccines induce opsonophagocyticantibody to both components.

II. Passive transfer. Passive immunization of naive mice with sera frommice immunized with 10 μg of COPS:FliC produced >80% protection againstlethal S. Enteritidis challenge, whereas mice receiving normal sera orPBS succumbed to infection, demonstrating that protection can bemediated in vivo by vaccine induced antibodies (TABLE 2).

TABLE 2 Efficacy of passive transfer into näive mice of sera from miceimmunized with COPS:FliC. Protection of mice from lethal challenge withwild-type S. Enteritidis R11^(a) Mortality Treatment (dead/total) PBS5/6 Normal serum 7/7 COPS:FliC serum  1/7^(b) ^(a)Mice challenged IPwith 5 × 10⁵ CFU ^(b)p = 0.005 compared to normal serum by 2-tailedFisher's exact test.

Development of opsonophagocytic antibody (OPA) assays—We have developedand validated a high-throughput flow cytometry based OPA uptake assayusing GFP-expressing PA (FIG. 10). We have also successfully adapted anopsonophagocytic assay that is widely used to evaluate pneumococcalcapsular polysaccharide vaccines (Romero-Steiner S et al., Clin DiagnLab Immunol. 1997; 4(4):415-422) to measure functional antibodieselicited by S. Typhimurium vaccines. This assay uses baby rabbitcomplement as a complement source and HL-60 cells as phagocytes. The OPAtiter is defined as the titer of sera that results in greater than 50%killing of bacteria following opsonophagocytosis. As shown in FIG. 11,OPA titers for sera from mice orally immunized with the live attenuatedS. Typhimurium vaccine CVD 1931 (ΔguaBA ΔclpX) were significantly higherthan for mice immunized with PBS.

Engineering bacteria so that large amounts of PA flagellin and Opolysaccharides (OPS) can be purified safely andeconomically—Large-scale fermentation using wild-type pathogenic KPbacteria to manufacture COPS constitutes a significant occupationalhealth hazard. The use of attenuated and avirulent strains from which topurify polysaccharide vaccine antigens markedly decreases these risks,and such a strategy is already being implemented for new generation S.Typhi Vi polysaccharide based vaccines (Micoli F et al., Vaccine. 2012;30(5):853-861). Precise deletions in select metabolic and virulencegenes of several GNB pathogens have resulted in attenuated strainsuseful as live oral vaccines (Tennant S M et al., Infect Immun. 2011;79(10):4175-4185; Tacket C O, Levine M M et al., Clin Infect Dis. 2007;45 Suppl 1:S20-23). We have experience in constructing such attenuatedvaccine strains and in demonstrating their clinical acceptability,safety and immunogenicity in animal models and in human clinical trials(Inaba S et al., Biopolymers. 2013; 99(1):63-72; Kotloff K L et al., HumVaccin. 2007; 3(6):268-275). We have had success using a guaBA mutation(Samant S et al., PLoS Pathog. 2008; 4(2):e37) as the primaryattenuating mutation in live attenuated Shigella vaccines where safetyhas been documented in clinical trials (Kotloff K L et al., Hum Vaccin.2007; 3(6):268-275). A Phase 1 clinical trial conducted at the CVD hasalso shown that S. Paratyphi A CVD 1902 (which possesses deletions inguaBA and clpX) was safe and well-tolerated in human volunteersincluding at the highest dosage levels tested (10¹⁰ CFU)(Levine MM.,Paper presented at: 8th International conference on typhoid fever andother invasive Salmonelloses 2013; Dhaka, Bangladesh). BecausePseudomonas aeruginosa expresses a solitary unipolar flagellum, thelevel of flagellin expression on Pseudomas aeruginosa is insufficientfor large scale production. Genetically engineered attenuated strainscan improve the safety of large-scale manufacture of Klebsiellapneumoniae OPS and can provide a means for enhanced Pseudomonasaeruginosa flagellin expression. Thus, we have created recombinantreagent strains that can be used to purify large amounts of Klebsiellapneumoniae OPS and PA flagellin.

Research Design for KP and PA strains—Genetically engineered Klebsiellapneumoniae reagent strains are created to improve occupational safetyfor large scale fermentation, and simplify and enhance OPS purificationand yields. GuaBA from K pneumoniae O1, O2, O3 and O5 strains is deletedusing lambda red recombination (Datsenko K A, Wanner B L., Proc NatlAcad Sci USA. Jun. 6 2000; 97(12):6640-6645). Capsule synthesis (cps)gene cluster is deleted from the four guaBA mutants. CPS mutation servestwo purposes: 1) It is a secondary independently attenuating mutationthat safeguards against the possibility of reversion to virulence; and2) purification of core-O polysaccharide will be simpler as there willbe no contaminating capsular polysaccharide.

The genes encoding PA flagellins FlaA and FlaB are cloned into pSEC10, ahighly stable low copy number plasmid, and then transform the plasmidsinto our S. Enteritidis reagent strain CVD 1947. The reagent strainsgrow in chemically defined minimal media and secrete large amounts of PAflagellin is confirmed by performing SDS-PAGE and western blots ofculture supernatant.

Reagents strains are grown in 5 L fermentation culture, as optimizationat this scale is generally translatable to larger volumes (e.g., 50L-1,000 L). KP reagent strain fermentation is optimzed with rich mediato supply an optimal environment for growth, making use of animalproduct free formulation to comply with FDA regulations for biologics.PA-Fla CVD 1947 expression vectors is grown in fully chemically definedminimal media to reduce the contaminant background, as the PA-Flaproduct will be in the supernatant. KP OPS and PA-Fla purification isconducted with previously optimized biochemical purification protocolsthat we developed for Salmonella COPS and FliC. Products are trackedthrough the process using standardized assays, and are verified to meetthe following release parameters (TABLE 3):

TABLE 3 Lot release parameters for purified KP COPS and PA flagellinsCOPS Flagellin Parameter limit (assay) limit (assay) Residual <1% (BCA)<1% (HPLC-SEC host cell protein with UV, SDS-PAGE) Residual nucleic acid<1% (A260 nm) <1% (Quant-IT) Residual endotoxin <150 EU/μg (LAL) <150EU/μg (LAL) Identity Conform to standards Expected size by (HPAEC-PAD,Western blot ELISA) Size/Weight HPLC-SEC HPLC-SEC with UV, with RISDS-PAGE

Construction of K. pneumoniae reagent strains—We genetically engineeredKlebsiella pneumoniae reagent strains to improve occupational safety forlarge scale fermentation, and simplify and enhance COPS purification andyields. We deleted guaBA from K. pneumoniae O1, O2, O3 and O5 strainsusing lambda red recombination. We also deleted the capsule synthesis(cps) gene cluster from the four guaBA mutants. CPS mutation will servetwo purposes: 1) It is a secondary independently attenuating mutationthat safeguards against the possibility of reversion to virulence; and2) purification of core-O polysaccharide will be simpler as there willbe no contaminating capsular polysaccharide.

We used lambda red recombination to delete guaBA (for attenuation) andthe capsule (cps) gene cluster from the following KP strains: B5055(O1:K2), 7380 (O2ab:K-), 390 (O3:K11) and 4425/51 (O5:K7). We havegenetically engineered the B5055 (O1) and 7380 (O2ab) Klebsiella strainsand have deleted guaBA and cps genes, as necessary. We have also createdthe 390 (O3) ΔguaBA mutant. See Table 4.

TABLE 4 CVD genetically engineered KP reagent strains Strain Parent O Kdesignation strain type type guaBA CPS Notes CVD 3000 B5055 1  2 − +Completed CVD 3001 B5055 1  2 − − Completed CVD 3010 7380 2 − −Naturally Completed Deficient CVD 3020 4425 5 57 − − In progress CVD3030 390 3 11 − − In progress

The primers used for the genetic engineering are shown in Table 5:

Name Target Purpose Primer sequences (5′→3′) guaBA_676_F B5055 ΔguaBAGGGTAGATGATCACCGGCA G (SEQ ID NO: 27) guaBA_688_R B5055 ΔguaBATGATTGGTCTGACTGGACGC (SEQ ID NO: 28) guaBA_155_R B5055 ΔguaBAGGAAGCCAGTGGGATCTGA C (SEQ ID NO: 29) guaBA_256_F B5055 ΔguaBACTGATCCAAACCTGGCCCAT (SEQ ID NO: 30) guamut_F B5055 ΔguaBAGGTCGACGGATCCCCGGAA TGGAGTAATCCCCGGCGTTA G (SEQ ID NO: 31) guamut_RB5055 ΔguaBA GAAGCAGCTCCAGCCTACA CGGGCAATATCTCGACCAG GG (SEQ ID NO: 32)guaA_R2 390- ΔguaBA CATACACCACGCGGGAGAT 4425/51- A (SEQ ID NO: 33) 7380guaA_mut_F2 390- ΔguaBA GGTCGACGGATCCCCGGAA 4425/51-TGCTAGCCGCGTTTTCGTGG 7380 AAGTG (SEQ ID NO: 34) guaB_F2 390- ΔguaBAGTCCTCCTCGTTCCCGCT 4425/51- (SEQ ID NO: 35) 7380 guaB_mut_R2 390- ΔguaBAGAAGCAGCTCCAGCCTACA 4425/51- CGAATTCCATCTTTACAGGC 7380GTTCGGT (SEQ ID NO: 36) wza_F B5055 Δwzabc GAGCCGACTCTAGGGTGGC 4425/51(SEQ ID NO: 37) wza wza_R B5055 Δwzabc GAAGCAGCTCCAGCCTACA 4425/51CTAATGTCACATCATCAGTA wza AATCAAAATTTG (SEQ ID NO: 38) K2_wzc_F B5055Δwzabc GAAGCAGCTCCAGCCTACA wzc CGTAATAGATATGTTATAGA GTTTGGAGGGGAG (SEQID NO: 39) K2_wzc_R B5055 Δwzabc TATTTAATTTCCCTCTTTCAT wzcCCTGTAATGTT (SEQ ID NO: 40) K11_wzc_F 390 Δwzabc GGTCGACGGATCCCCGGAA wzcTTGTTTCAAGATTATATATTT CGATGCCTAATG (SEQ ID NO: 41) K11_wzc_R 390 ΔwzabcTCCTTAGTATAAAGTTGAGA wzc GATTTCTGATTC (SEQ ID NO: 42) K57_wzc_F 4425/51Δwzabc GGTCGACGGATCCCCGGAA wzc TGAATCGGATGATATCGATTTAGGTAAAATTGT (SEQ ID NO: 43) K57_wzc_R 4425/51 ΔwzabcGCTAATAGCTTTCAAACGAC wzc TTATATAGGTTA (SEQ  ID NO: 44) P1 pKD13- Kan-GTGTAGGCTGGAGCTGCTT kan cassette C (SEQ ID NO: 45) P4 pKD13- Kan-ATTCCGGGGATCCGTCGACC kan cassette (SEQ ID NO: 46)

Deletion of guaBA from K. pneumoniae B5055—DNA was first purified fromB5055 with the Qiagen DNEasy Blood and Tissue kit according to themanufacturer's protocol. DNA upstream of guaA was amplified using thefollowing primers that produce a 688 bp DNA fragment (KP_guamut_F:5′-GGTCGACGGATCCCCGGAATGGAGTAATCCCCGGCGTTAG-3′ (SEQ ID NO:31); KPguaBA_688_R: 5′-TGATTGGTCTGACTGGACGC-3′ (SEQ ID NO:28)). DNA downstreamof guaB was amplified using primers that produce a 676 bp DNA fragment(KP guaBA_676_F: 5′-GGGTAGATGATCACCGGCAG-3′ (SEQ ID NO:27); KP_guamut_R:5′-GAAGCAGCTCCAGCCTACACGGGCAATATCTCGACCAGGG-3′ (SEQ ID NO:32)). PCRamplification of the guaA/guaB flanking regions was conducted using Ventpolymerase. PCR products were electrophoresed on a 1% agarose gel andextracted and purified with a Qiagen Gel extraction kit according to themanufacturer's protocol. The PCR products were combined in anoverlapping PCR reaction using a Kan cassette amplified from pKD13 asdescribed by Datsenko and Wanner. The PCR product of ˜2.4 kb was gelextracted and amplified with guaBA_676_F/guaBA_688_R beforetransformation. Electrocompetent B5055 cells were transformed byelectroporation with pKD46. Electrocompetent cells of K. pneumoniaeB5055 expressing lambda red recombinase were prepared and electroporatedwith the 2.4 kb PCR product. Kanamycin resistant colonies were selectedand screened for integration of the Kanamycin resistance cassette. TheKanamycin resistance cassette was subsequently deleted using pCP20 thatremoves the cassette via the FRT sites present in the sequence. Toremove pCP20, cells were grown at 42° C. and tested after each passagefor loss of Carbenicillin or Chloramphenicol resistance.

Deletion of capsule genes from K. pneumoniae B5055—The genes encodingcapsule synthesis in K. pneumoniae B5055 were also deleted using lambdared recombination. DNA downstream of wza was amplified using thefollowing primers that produce a 600 bp DNA fragment (wza_F:5′-GAGCCGACTCTAGGGTGGC-3′ (SEQ ID NO:37); wza_R:5′-GAAGCAGCTCCAGCCTACACTAATGTCACATCATCAGTAAAT CAAAATTTG-3′ (SEQ IDNO:38)). Primers for the other flank amplify a region inside wzc itselfsince it is specific for the capsule type while the surrounding regionsare highly variable between different capsule types. The primers(K2_wzc_F: 5′-GGTCGA CGGA TCCCCGGAA TGTAATAGATATGTTATAGAGTTTGGAGGGGAG-3′(SEQ ID NO:39); K2_wzc_R: 5′-TATTTAATTTCCCTCTTTCATCCTGTAATGTT-3′ (SEQ IDNO:40)) produced a 600 bp fragment. The same procedure as used for theguaBA mutagenesis were used.

Schematic diagrams of the guaBA and wzabc genetic regions of K.pneumoniae are shown in FIG. 12. Schematic diagrams of the DNA removedduring mutagenesis of guaBA and wzabc from K. pneumoniae are shown inFIG. 13. Mutagenesis was verified by PCR and sequencing upstream anddownstream of the deletion is shown in FIG. 14.

The capsule deletion was assessed by India Ink staining and microscopicobservation of the parental and mutant strain. The K. pneumoniae B5055ΔguaBA Δwzabc strain showed no evidence of capsule whereas the wild-typestrain was capsule positive.

We have confirmed the guanine auxotrophy phenotype by growing therecombinant strains on minimal media containing or lacking guanine (FIG.15). We have shown that guanine must be supplied for growth of the KPΔguaBA mutants. Verification of attenuation—KP O1:K2 strains are highlyvirulent for mice but most other serotypes that are human pathogens havebeen found to be avirulent in mice. To confirm that the CVD 3001 reagentstrain (B5055 ΔguaBA Δwzabc) is attenuated, we tested this mutant inmice and showed that the intraperitoneal 50% lethal dose is higher thanthe wild-type parental strain (Table 7). LD5₀ analysis was conductedusing 5 CD-1 mice per group injected IP with 10-fold dilutions ofwild-type KP and the candidate engineered attenuated derivative.

TABLE 6 Verification of attenuation of KP reagent strain in vivo. Strain50% lethal dose Wild-type B5055 5.34 × 10³ CFU B5055 ΔguaBA >10⁸ CFUΔwzabc (CVD 3001)

Construction of recombinant S. Enteritidis that express Type A and Bflagella from P. aeruginosa—We cloned flaA from P. aeruginosa PAK whichencodes Type A flagella (40 kDa) into pSEC10, a low copy number highlystable plasmid. We also cloned flaB from P. aeruginosa PAO1 whichencodes Type B flagella (52 kDa) into pSEC10. The recombinant plasmidswere transformed separately into CVD 1947 (S. Enteritidis R11 ΔguaBAΔclpP ΔfliD ΔfliC) to create reagent strains capable of expressing largeamounts of Type A or B flagellin. Mutagenesis was verified by PCR andsequencing upstream and downstream of the deletion. Secretion of Type Aor B flagella was verified by SDS-PAGE.

The fliC gene was amplified from P. aeruginosa PAK using primersPAK_fliC_F and PAK_fliC_R and cloned into pSEC10 so that it is expressedusing the PompC promoter. Likewise, the fliC gene was amplified from P.aeruginosa PAO1 using primers PAO1_fliC_F and PAO1_fliC_R and clonedinto pSEC10 so that it is expressed using the PompC promoter. Primersused for cloning are shown in Table 4. Schematic diagrams of theresultant plasmids pSEC10-flaA and pSEC10-flaB are shown in FIGS. 16 and17, respectively.

TABLE 7 Primers used for cloning of P. aeruginosa fliC genes in pSEC10.Restriction Name Strain site Sequence (5′-3′) PAK_fliC_ PAK BamHITATCTAGOATCCATGOCCT F TGACCGTCAACAC (SEQ ID NO: 47) PAK_fliC_ PAK NheICTAAGTGCTAGCAAGCTT R AGCGCAGCAGGCT (SEQ ID NO: 48) PAO1_fliC_ PAO1 BamHIACTTGCGGATCCATGGCC F CTTACAGTCAAACG SEQ ID NO: 49) PAO1_fliC_ PAO1 NheIATTAGCGCTAGCCGTGAG R TGACCGTTCCCG (SEQ ID NO: 50)

Construction of the reagent strain S. Enteritidis CVD1947—We previouslyused Salmonella Enteritidis CVD 1943 (R11 ΔguaBA ΔclpP ΔfliD) to expresslarge amounts of flagellin into the supernatant. We geneticallyengineered this strain so that it no longer expresses native fliC. Theobjective is to use this strain to express exogenous fliC from a plasmidand which is secreted into the supernatant. We used lambda redrecombination to delete the fliC gene. To ensure transcription ofdownstream genes after deletion of fliC in CVD 1943, the kanamycincassette from pKD4 was used since it allows conservation of multiplepromoter sites in the scar region after removing the kanamycin cassettefrom the genome. The primers shown in Table 5 were used to create aconstruct by overlapping PCR containing the Kanamycin cassette flankedby DNA upstream and downstream of fliC. Primers R11_fliC_up_F3 andR11_all_up_R3 amplify a 259 bp fragment upstream of fliC.R11_fliC_dwn_F3 and R11_fliC_dwn_R3 amplify a 301 bp fragment downstreamof fliC. The fliC gene was subsequently deleted using lambda redrecombination.

TABLE 8 Primers used for mutagenesis of Salmonella Enteritidis CVD1947.Name Target Sequence (5′-3′) P1 pKD4 GTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 51)  P2 pKD4 CATATGAATATCCTCCTTA (SEQ ID NO: 52) R11_filC_R11 CCATGCCATCTTCCTTTCG up_F3 (SEQ ID NO: 53) R11_all_ R11 (P1GAAGCAGCTCCAGCCTACACG up_F3 cpt) ATCTTTTCCTTATCAATTACAACTTG (SEQ ID NO: 54) R11_filC_ R11 (P2 TAAGGAGGATATTCATATGATC down_F3cpt) CGGCGATTGATTCAC (SEQ ID NO: 55) R11_filC_ R11 TGGTAATTTAATCTCCCCCCAdown_R3 (SEQ ID NO: 56)

We verified the deletion of fliC in CVD 1947 by sequencing the deletion.The entire fliC gene was deleted (FIG. 18).

The pSEC10-flaA and pSEC10-flaB plasmids were transformed into S.Enteritidis CVD 1947. We confirmed that CVD1947 (pSEC10-flaA) and CVD1947 (pSEC10-flaB) can express FlaA and FlaB in the supernatant wherethey demonstrated the approximate predicted molecular weight of ˜45 kDaand ˜50 kDa respectively by SDS-PAGE and coomassie analysis (FIG. 19).Secreted recombinant FlaA expressed in CVD 1947 was also recognized bywestern blot with polyclonal sera from mice immunized with purifiednative FlaA obtained from P. aeruginosa strain PAK (FIG. 20).

Purification and characterization of Klebsiella pneumoniae O1O-polysaccharide (OPS)—Recombinant K. pneumoniae strain CVD3001 wasgrown to stationary phase by overnight growth in shaking culture at 37°C. in fully chemically defined media supplemented with guanine. OPS wasextracted from the bulk growth culture by two different methods. In thefirst method, OPS was released from the core PS KDO by reduction of theculture pH to ˜3.7 with acetic acid and incubation at 100° C. for 4hours. In the second method, the culture was brought to pH ˜3.7 withacetic acid and incubated with 200 mg/L sodium nitrite for 6 hours at 4°C. to release the OPS by nitrous acid deamination. Following OPSrelease, cells and insoluble debris were removed by centrifugation andclarification through a 0.45 um filter. Extraction by either methodyielded OPS molecules of similar size that could be distinguished fromresidual contaminants in the post-hydrolysis supernatant byhigh-performance liquid size-exclusion chromatography (HPLC-SEC)analysis with detection by refractive index (RI) (FIG. 21, 22). The OPSwas purified from residual soluble contaminants by sequential stepsinvolving 30 kDa molecular weight cutoff (MWCO) tangential flowfiltration (TFF), anion-exchange chromatography, and ammonium sulfateprecipitation. The purified material was concentrated and diafilteredinto water by 10 kDa MWCO TFF. Analysis of the final-purified andin-process material by HPLC-SEC/RI demonstrated a single major molecularweight OPS species that was retained throughout the purification process(FIG. 21, 22).

The identity of the final purified O1 OPS was accomplished bydepolymerization with 2M Trifluoroacetic acid and analysis of themonosaccharide constituents by high-performance anion-exchangechromatography with pulsed amperometric detection (HPAEC-PAD) (FIG. 23).Monosaccharide composition analyses revealed that the OPS was comprisedprimarily of galactose with a minor N-acetyl-glucosamine peak detected.This is consistent with the published chemical structure of O1 OPS thatis comprised entirely of galactose with a terminal N-acetyl-glucosamineresidue present at the reducing end adjacent to the KDO, that is theexpected site of hydrolysis by our extraction method (FIG. 1)(Vinogradov et al., J Biol. Chem. 2002; 277:25070-25081).

Expression and purification of Pseudomas aeruginosa flagellin—Expressionof rFlaA for subsequent purification was accomplished by growing CVD1947 containing pSEC10_rFlaA in fully defined chemical mediasupplemented with guanine and kanamycin at 37° C. under shakingconditions to mid-log phase. The culture supernatant containing thesecreted rFlaA was clarified from cells by centrifugation and filtrationwith a 0.45 um filter. rFlaA was then purified from the clarifiedculture supernatant using sequential cation- and anion-exchange membranechromatography steps as described (Simon Ret al., Protein Expr. Purif.2014; 102:1-7). SDS-PAGE and coomassie analysis of the final purifiedproduct confirmed a single ˜45 kDa band (FIG. 24).

Conjugation of Klebsiella pneumoniae O1 OPS with recombinant Pseudomonasaeruginosa FlaA—OPS was activated with CDAP at pH 9, added directly topurified recombinant FlaA, and incubated overnight at 4° C. Conjugationwas assessed by HPLC-SEC (FIG. 25), where a shift in size was seen tohigher molecular weight species after linkage. Unconjugated OPS andrFlaA produced distinct peaks at ˜10 minutes and 10.8 minutesrespectively, whereas the conjugated material produced a sharp peak at˜5.5 minutes that represents the column void volume (>650 kDa) with alarge trailing tail of smaller conjugates persisting till ˜9 minutes.This indicates a heterogenous conjugate population comprised by largeand very large molecular weight species. Due to the very high molecularweight of the conjugate seen by HPLC-SEC, it is likely that much of theconjugated material is too large to enter an SDS-PAGE gel. Nevertheless,SDS-PAGE analysis with coomassie staining (FIG. 26A) confirmed the shiftto a heterogeneous mix of higher molecular weight species seen by thesmear above the level of remaining unconjugated protein. Western blotanalysis performed on the purified flagellin and KP-O1:PA-rFlaAconjugate with polyclonal mouse anti-sera raised against native FlaA(FIG. 26B) confirmed identity of the conjugated material seen in thematerial that was of sufficient size to enter the gel matrix.

Dot blot analysis of the conjugate and unconjugated polysaccharideconfirmed reactivity of the conjugate with sera from mice administeredCVD 3001 (KP O1 reagent strain deleted for guaBA and capsule synthesisgenes). A robust signal was seen for the conjugate, whereas thepolysaccharide did not bind the membrane as no protein component waspresent that is required for binding, thus confirming that conjugatedsaccharide was reactive with the anti-O1 antibodies (FIG. 27).

Constructing and assessing in rodents KP:PA conjugates with differentcombinations and chemistries in monovalent and quadrivalentformulations—Klebsiella pneumoniae LPS and Pseudomas aeruginosaflagellins have been demonstrated conclusively as protective against thecognate pathogens expressing these antigens; however, there are noreports of vaccination approaches to elicit protective immunity to bothof these molecules in a single formulation. As LPS is unacceptablyreactogenic, and isolated OPS molecules are generally non-immunogenic, aconjugate vaccine approach is warranted. Remarkably, our literaturesearch revealed only two published reports of KP COPS conjugates (withTT or KP OMPs), and while protection was documented, ELISA antibodytiters and boost responses were not assessed (Chhibber S, Indian J ExpBiol. 2005; 43(1):40-45; Chhibber S, Vaccine. 1995; 13(2):179-184).

Flagellins have been found as effective carrier proteins, however, themajority of licensed conjugate vaccines use established vaccine proteinsas carriers (e.g., TT, diphtheria toxoid)(Knuf M, Vaccine. 2011;29(31):4881-4890), that are already administered separately as vaccineantigens, and for which immunity from the carrier protein is not a basisfor licensure (Knuf M, Vaccine. 2011; 29(31):4881-4890). The exceptionis the GlaxoSmithKline 10-valent pneumococcal conjugate vaccineSynflorix™ that uses Haemophilus influenzae protein D as a carrierprotein to extend protection against non-typeable H. influenzae acuteotitis media (Forsgren A et al., Clin Infect Dis. 2008; 46(5):726-731;Prymula R, Schuerman L, Expert Rev Vaccines. 2009; 8(11):1479-1500). Twomajor challenges are thus addressed by our development approach. First,we will confirm induction of functional immunity by both thepolysaccharide hapten and protein carrier Immunogenicity and functionalefficacy for OPS and Fla induced antibodies will likely be influenced byphysicochemical conjugate structure. The size, structure, and level ofsolvent accessible protein and polysaccharide residues have beendocumented to influence coupling site preference in glycoconjugates(Bardotti A et al., Vaccine. 2008; 26(18):2284-2296); this could affectfunctional immunogenicity if important protective epitopes are thepreferential sites of linkage. A minimal level of vicinialpolysaccharide epitopes is necessary to cross-link B-cell receptors(BCR). We have found that conjugation of equal weights of COPS andflagellin produces linkage ratios that are immunogenic (Simon R, InfectImmun. 2011; 79(10):4240-4249; Raphael Simon J Y W et al., PLOS ONE.2013; 8(5): e64680). Lattice type conjugates provide larger surfaces forBCR cross-linking, however, CDAP activation also alters polysaccharidelinkage point epitopes. End-linkage with oxime chemistry does not alterPS epitopes, but forms smaller conjugates. By screening COPS conjugatesmade with different chemistries and PA Fla types, we expect to identifyoptimal monovalent conjugate formulations. Secondly, possibleinterference between individual components is a recognized pitfall ofmultivalent vaccine formulations. Thus, it will be confirmed that theimmunogenicity and efficacy of individual component OPS and flagellinantigens are preserved when combined into a quadrivalent formulation. Byidentifying in animals optimally immunogenic and protective monovalentconjugate architectures and confirming immunogenicity whenco-formulated, effective quadrivalent formulations will be produced.

Prior to undertaking a quadrivalent conjugate screen, proof-of-conceptwill first be established for a single candidate monovalent antigenKP-OPS:PA-Fla conjugate type synthesized with material purified fromshake flask cultures. Accordingly, we will construct candidateconjugates of KP O1 OPS with type A PA flagellin, as both of these typeshave been reported extensively as protective vaccine antigens using wellcharacterized challenge strains and infection models. We will generate asun-type KP-O1-OPS:PA-FlaA conjugate using oxime chemistry as well as alattice type conjugate using CDAP, and immunize mice (n=30/group) 3times at 28 day intervals with PBS, KP-O1-OPS:PA-FlaA conjugates, or O1OPS alone or admixed with FlaA. We will assess the kinetics andinduction of anti-LPS and anti-Fla antibody responses by measuring thelevel of vaccine induced IgG antibodies in sera before immunization and21 days after each vaccine dose by ELISA with purified antigens, and bymeasuring functional activity using motility inhibition and OPA assays.We will determine whether KP-O1-OPS:PA-FlaA conjugates are protectiveagainst infection, by challenging IP with KP (n=15/group) or in burnwound infections with PA (n=15/group), as protection mediated by theseKP and PA antigens is best established with these challenge routes. Forchallenge studies, we will use PA PAK that is a type Aflagellin-expressing isolate or KP B5055 that is an encapsulated O1:K2isolate, as both have been used extensively as challenge strains forvaccine studies in mice (FIG. 28).

The conjugate type demonstrating the best immunogenicity and protectionin will be assessed for protection against Klebsiella pneumoniae (KP)B5055 or Pseudomas aeruginosa (PA) PAK administered by several routes ofinfection (burn, myositis, punch wound, or IP septicemia). Mice(n=120/group) will be immunized 3 times at 28 day intervals with PBS orKP-O1-OPS and PA FlaA conjugated or admixed. Preimmune sera and seraobtained 21 days after the final vaccination will be assessed forfunctional and binding antibodies with homologous antigens and strains.Mice (n=15/group) will be infected (wound and IP) with PA or KP (FIG.29).

We will generate 16 different candidate conjugates by linking theindividual KP OPS serotypes using CDAP or oxime chemistry to FlaA orFlaB, using material obtained from fermentation cultures. The twomonovalent KP-O1-OPS:PA-FlaA CDAP and oxime conjugate types createdabove will be included for confirmation of previous results. Thisconjugate panel will be tested individually in mice by immunizing with 3doses spaced 28 days apart (n=30/group). Pre-immune sera and sera taken21 days after the final dose will be assessed for homologous anti-LPSand anti-flagellin IgG levels by ELISA and functional antibodies by OPAor motility inhibition assays. We will screen for functional efficacy ofvaccine-elicited antibodies in vivo by measuring protection against IPinfection with the homologous KP O type expressing strain (O1: B5055;O2, O3 and O5: recombinant mouse virulent strains that we will generate;n=15/group), and burn wounds with the homologous flagellin expressing PAstrain (n=15/group)(FIG. 30).

A single monovalent conjugate from each OPS type will subsequently beselected for inclusion in a quadrivalent formulation based on thefollowing ranked criteria: 1) anti-LPS IgG levels and KP OPA antibodytiters, 2) anti-flagellin IgG and functional anti-PA antibody titers, 3)protective efficacy, 4) regulatory and manufacturing considerations(yield, ease of synthesis, epitope preservation, and regulatoryprecedent). We place anti-OPS responses as more critical thananti-flagellin responses in our go/no-go decision tree, as moderateanti-flagellin immune responses could be compensated for in a finalformulation by the inclusion of multiple conjugates that utilize thesame flagellin protein carrier. The final quadrivalent conjugateformulation will include minimally at least one conjugate made with eachflagellin type, and would be anticipated to impart high IgG antibodylevels to both flagellin types. We recognize as well that mouseprotection studies may not always fully recapitulate the truepathogenicity of a given bacterial strain in humans, nor fullyapproximate the mechanisms of protective immunity. This is particularlytrue for KP, as numerous examples exist of human clinical isolates thatdemonstrate poor pathogenicity in mice (Struve C, Krogfelt K A, EnvironMicrobiol. 2004; 6(6):584-590; Simoons-Smit A M, J Med Microbiol. 1984;17(1):67-77; Yu V L, Emerg Infect Dis. 2007; 13(7):986-993). Hence,while protection is one important measure of down-selection and isexpected to approximate vaccine performance in humans, we place greatercredence on the capacity to induce robust seroconversion levels and hightiters in our chosen functional antibody assays, as these are theanticipated correlates and mechanisms of protection for humans.

To confirm that the specific immune responses to FlaA and FlaB aremaintained when co-formulated, mice will be immunized 3 times at 28 dayintervals with monovalent (n=15/group) and bivalent (n=30/group)flagellin preparations. Levels of IgG and functional titers for thehomologous Ha types will be determined in pre-immune sera and sera taken21 days after the final dose. Protection will be assessed against burninfection with homologous Fla expressing PA strains (n=15/group)(FIG.31).

An assay will be conducted to confirm that the humoral responses andprotective efficacy of the 4 down-selected monovalent COPS and flagellinconjugate vaccine components are maintained when administered as amultivalent vaccine formulation. Mice (n=240/group) will be immunized 3times at 28 day intervals with PBS or the quadrivalent formulation, or 2individual monovalent conjugates (n=120/group)(FIG. 32). For monovalentconjugates, we will include a KP O1 conjugate for comparison withprevious proof-of-concept wound protection results, the second selectedconjugate will be of a different OPS type and flagellin type, and willconfirm the general protective efficacy against wound infections formonovalent and quadrivalent KP-OPS:PA-Fla conjugates. Sera obtainedprior to immunization and 21 days after the final dose will be assessedfor anti-LPS and anti-flagellin antibodies. We will also assessfunctional opsonophagocytic titers as well as inhibition of PA motilitywith homologous antigen pathogens. The protective efficacy ofquadrivalent-relative to monovalent—vaccines to prevent invasive andwound infections will be determined using the IP, myositis, burn woundor punch-biopsy models and homologous KP O-type pathogens (n=15/group)or the homologous flagellin type expressing PA (PAK or PAO1)(n=15/group)(FIG. 32).

We will assess the utility of the quadrivalent conjugate formulation togenerate antibody preparations that can be used therapeutically as IVIG.For this, rabbits will be hyper-immunized with quadrivalent vaccine andpooled sera will be prepared for use in passive transfer studies inmice. The level of anti-LPS and anti-flagellin IgG in rabbit sera willbe determined by ELISA, as well as functional antibody titers by OPA andmotility inhibition assays. Dosage levels will be approximated to theantibody titer induced by active immunization in mice. Naive mice(n=30/group) will be intravenously administered immune sera, normal(unimmunized) rabbit sera (N.S.), or PBS, followed by IP or burninfection 2-4 hours later with KP B5055 or PA PAK, respectively (FIG.33).

Construction of conjugate vaccines—Random linked lattice-type conjugateswill be generated as described (Simon R, Infect Immun. 2011;79(10):4240-4249; Shafer D E et al., Vaccine. 2000; 18(13):1273-1281;Lees A et al., Vaccine. 1996; 14(3):190-198) with direct conjugation toprotein lysines by activation of COPS with CDAP, and reacting with anequal ratio by weight of flagellin protein at pH 9-10. End-linkedsun-type conjugates will be prepared with thioether oxime chemistry(Lees A et al., Vaccine. 2006; 24(6):716-729; Kubler-Kielb J., MethodsMol Biol. 2011; 751:317-327) by derivatizing the COPS KDO reducing endcarbonyl group with a diamin000xy cysteamine linker and reacting at atwo-fold polysaccharide to protein weight with sulfo-GMBS attached atflagellin protein lysines. Conjugation will be confirmed by SDS-PAGEwith Coomassie (Thermo) staining for protein and Pro-Q (LifeTechnologies) staining for polysaccharide, and by HPLC-SEC for size.Unreacted conjugation chemicals and conjugate components will be removedas described by size-exclusion chromatography with SUPERDEX 200 (GE) andanion exchange membrane chromatography (Sartorius)(Simon R, InfectImmun. 2011; 79(10):4240-4249). Final levels of polysaccharide andprotein in conjugates will be determined by resorcinol assay (Monsigny Met al., Anal Biochem. 1988; 175(2):525-530) and BCA assay (Thermo)respectively, with unconjugated standards. Conjugates will be stored at4° C. in 20 mM Tris pH 7 until use. We will confirm loss of TLRSactivity using the IL-8 release assay as described (Turley C B et al.,Vaccine. 2011; 29(32):5145-5152; Taylor D N et al., Vaccine. 2011;29(31):4897-4902; Liu G et al., PLoS One. 2011; 6(6):e20928; Song L etal., Vaccine. 2009; 27(42):5875-5884).

Immunization and serological measurements—Six- to 8-week-old femaleoutbred (CD-1/ICR) mice will be immunized intramuscularly on threeoccasions at 28 day intervals with either PBS, 2.5 μg of unconjugatedflagellin or OPS, 2.5 μg by polysaccharide weight for monovalentconjugates, or 10 μg of total polysaccharide in a quadrivalent conjugateformulation. Sera will be obtained via the retroorbital plexus.Anti-flagellin and anti-OPS serum IgG titers will be assessed by ELISAas described (Simon R, Infect Immun. 2011; 79(10):4240-4249).

Construction of mouse virulent challenge strains—K. pneumoniae O1:K2strains are highly virulent for mice but most other serotypes, that arehuman pathogens, have been found to be avirulent in mice. In fact,virulence in mice is attributed to the K2 capsule. Kabha et al. (Kabha Ket al., Infect Immun. 1995; 63(3):847-852) have shown that when the cpsgenes that encode the K2 capsule are cloned into an avirulent KP strain,the recombinant strain shows increased virulence for mice, albeit at alevel intermediate between the fully virulent and avirulent strains.First, we will determine the virulence for our O2, O3 and O5 strains inmice by the intraperitoneal route, as this is a good test for invasivepathogenicity. If we find an LD₅₀<10⁶ CFU, we will not manipulate thestrains and will use the wild-type strains in challenge experiments. Ifthe LD₅₀ is >10⁶ CFU, we will clone the cps gene cluster that encodes K2capsule into the putative avirulent O2, O3 and O5 strains and confirmpathogenicity in mice. We will confirm successful genetic engineering byPCR and sequencing, and the expected phenotype by western blot usingpolyclonal K2 antisera (Cryz S J, Jr., J Infect Dis. 1991;163(5):1055-1061). For PA, we will use the well characterized PAK (FlaA)and PA:O1 (FlaB) that are pathogenic in mice.

Preparation of bacteria for functional antibody assays and challengeexperiments—For challenge experiments, bacteria will be prepared asdescribed in the art (Cryz S J et al., J Lab Clin Med. 1986;108(3):182-189). Bacteria are streaked on agar for single colonyisolation to ensure purity. Three to 5 colonies are inoculated into richmedia liquid broth and grown to mid-log phase under shaking aerationconditions at 37° C. Bacterial cultures are then pelleted, washed in PBSand adjusted to 0.3 OD₆₀₀ that we have previously determined representsapproximately 1×10⁸ CFU/ml. For OPA assays and motility assays, andchallenge experiments with PA, we will use the established PAK and PAO1strains. Expression of CPS when KP are grown in broth culture has beendemonstrated as growth-phase dependent (Favre-Bonte S, Infect Immun.1999; 67(2):554-561; Mengistu Y et al., J Appl Bacteriol. 1994;76(5):424-430), and different CPS types may display different levels ofcell coverage in broth cultures. To enable easier handling of KP, and tobetter standardize OPS accessibility for functional OPA assays designedto down-select conjugates, we will use the KP AguaBA Acps O1, O2, O3 andO5 strains as target strains. These recombinant strains will be safer tohandle from an occupational health and safety stand-point and will lackthe mucoid nature of the wild-type strains. For challenge experiments,we will use wild-type capsulated strains. For all challenge strains tobe used in animal experiments, in order to attain 100% attack rate incontrols, we will independently determine the LD₅₀ for each route ofinfection, and base our challenge dose on the required multiple of LD₅₀necessary to attain an LD₁₀₀.

Opsonophagocytic assays. In order to measure enhancement of bacterialuptake, a flow cytometric assay is instituted. Briefly, human PMNs aremixed with a GFP-expressing target bacterial strain in the presence orabsence of antibody. The bacteria are spun onto the PMNs at 4° C.,incubated for 15 min at 37° C., washed again and resuspended in mediumcontaining gentamicin (50 μg/ml) for 15 min, washed and then resuspendedin PBS for flow cytometric analysis with gating on the PMNs by forwardand side scatter. The uptake of the GFP-labelled bacteria is thendetermined. Either GFP-expressing PA or KP is added to the PMNs in thepresence and absence of pre- and post-immune mouse sera from each of theconjugate vaccines. As a positive control, PMNs and bacteria are mixedwith IVIG enriched in antibodies to PA and KP as we previously described(Ramachandran G et al., J Infect Dis. 2013; 207(12):1869-1877). Asfurther controls, the PMNs and bacteria are incubated at 4° C. that willinhibit bacterial uptake. Mixtures are incubated at 37° C. for 30 min,washed 3 times and analyzed by flow cytometry in the Flow Cytometry Coreat the CVD. The uptake and killing measured by this high throughputsystem will be confirmed in a HL-60 cell assay using live colony counts.Opsonophagocytic killing will be evaluated using the pneumococcal OPAassay that is accepted by the FDA. 20 μl (˜700-1000 CFU) of KP or PAgrown to log phase are combined with two-fold serial dilutions of serumand incubate at 37° C. for 15 min in a 5% CO₂ incubator to allow theantibody to opsonize the bacteria. Then, 10 μl of BRC and 40 μl ofdifferentiated HL-60 cells are added (4×10⁵ cells/well) and incubated at37° C. for 45 min. The negative control contains bacteria, HL-60 cellsand complement only. OPA titer is defined as the reciprocal of thehighest serum dilution that produced >50% killing in relation to thekilling observed for control containing only bacteria, HL-60 cells andcomplement (no serum).

Motility inhibition assays—The ability of conjugate vaccine antisera toblock motility of PA expressing Type A and B flagella is determined. Asdescribed by Brett et al (Brett P J et al., Infect Immun. 1994;62(5):1914-1919), two-fold dilutions of antisera with motility mediumare mixed and the agar is allowed to set. Motility assays are performedby stabbing the agar with PA and incubating overnight. The zone ofmotility is measured the next day.

Hyper-immunization of rabbits to obtain IVIG—New Zealand White rabbits(n=2/group) are immunized intramuscularly 4 times at 2-week intervalswith the quadrivalent conjugate containing 10 μg total polysaccharide.One control rabbit is immunized with four doses of PBS to obtain normalrabbit serum. Pooled sera taken 28 days after the last immunization isassessed for titers of anti-COPS and anti-Fla IgG by end-point dilutionELISA with purified antigens and functional antibody with OPA andmotility inhibition assays. Sera is heat inactivated prior to use infunctional assays and passive transfer, to ablate potential serumbactericidal killing by rabbit complement.

Passive transfer immunization—For passive transfer protection assays,mice are administered 0.2 ml heat-inactivated rabbit sera intravenouslyby the tail vein at the approximate total EU/mouse obtained with activeimmunization, and infected 2-4 hours later with a lethal dose of KP orPA.

Mouse peritonitis challenge—Mice are infected IP with either PA or KP(at the minimal reliable LD₁₀₀) and weight and survival (or moribundity)is followed for 14 days. Mouse myositis infection—The mouse thigh muscleinfection model has been used extensively to assess antimicrobial agents(Fantin B et al., Antimicrob Agents Chemother. 1991; 35(7):1413-1422).Following immunization at days 0, 14 and 28 with either monovalentvaccine or PBS, mice are administered an LD₁₀₀ dose of KP or PAsuspended in 100 μl of PBS into the thigh muscle of the mouse and weightand survival (or moribundity) followed for 14 days.

Burned mouse model—Under anesthesia and analgesia, mice are subjected toa nonlethal thermal injury with the burned mouse model described (Cryz SJ, Jr., Infect Immun. 1984; 45(1):139-142; Stieritz D D, Holder I A. JInfect Dis. 1975; 131(6):688-691; Neely A N et al., J Burn Care Rehabil.2002; 23(5):333-340; Horzempa J et al., Clin Vaccine Immunol. 2008;15(4):590-597). A heat-resistant polymer card template with a 1 by 1.5inch opening is pressed firmly against the shaven back. Ethanol isevenly spread over the area of the back outlined by the window, ignitedwith a lit cotton swab, and allowed to burn for precisely 10 seconds andextinguished Immediately after the burn, the mice are given 0.5 ml ofsterile normal saline intraperitoneally as fluid replacement therapy.This method reproducibly yields a 12-15% total body surface areafull-thickness burn which, by itself, is nonlethal. Burned mice arechallenged with a subeschar LD₁₀₀ injection of either PA or KP. Mice areobserved daily for 14 days during which time morbidity and mortalitywill be recorded. As controls, a separate “bystander group” is included,that will include burned but uninfected mice inoculated with salinealone.

Mouse punch-biopsy model—Mice are anesthetized by intraperitonealinjection of 100-150 μl of ketamine (100 mg/kg)/xylazine (10 mg/kg)prior to performing a dermal wounding procedure. After anesthesia, thedorsum of the mouse is shaved with an electric razor. The surgery areais sterilized with iodine and 70% alcohol. A full-thickness, excisionaldermal wound is made on the back of each mouse with a 6 mm sterilebiopsy punch, and a LD₁₀₀ bacterial dose in 25 μl will be inoculated onthe wound site. Other groups of mice are wounded, but not inoculatedwith bacteria, and serve as negative controls. Wounded mice are observedfor 7 days, monitoring for mortality and moribundity. As alternativeendpoints prior to mortality, mice are evaluated for wound size, grosspathology, weight, and colonization by excising a 2-4 mm tissue punchbiopsy from the wound bed to determine CFU/g. All challenge experimentsare conducted without the use of an immunosuppressive agent, as wepresume functional activity of professional phagocytes to be a keymechanism of vaccine-mediated protection.

Statistical power—A titer of ≧4-fold over pre-immune levels for ELISAand OPA assays, or ≧50% reduction in PA motility zone, will be assignedas the threshold for seroconversion. For comparison of seroconversionrates between monovalent vaccines, if the true underlying rate one typeof conjugate achieves is 85% or greater, with 30 mice/group we will have94% power to detect a significant difference, if seroconversion in micegetting the other type of conjugate is 40% or less (Fisher's exact test,α=0.025, 1 tail). Power will be 49% if the seroconversion rate in micegetting the other type of conjugate is 60%. For comparisons ofmonovalent and quadrivalent formulations, if the true seroconversionrate is 80% for each formulation, with 60 mice per group, we will have77% power to find non-inferiority using a non-inferiority margin of 20%(i.e., to obtain a 2-sided 95% confidence interval, by a likelihoodscore method, for the absolute difference of monovalent-quadrivalentwith upper limit ≦20%). Challenging with an LD₁₀₀ of wild-type KP or PAis expected to cause 100% mortality in unimmunized mice. Thus, ifmortality for a conjugate vaccine is reduced by 50% or greater, with 15mice/group we will have 94% power to detect a significant difference(Fisher's exact test, α=0.025, 1 tail). If mortality is 10% for onevaccine and 70% for another vaccine, with 15 mice/group, we will have89% power to find a significant difference between conjugates (Fisher'sexact test, α=0.025, 1 tail). If mortality is 20% for one vaccine and70% for the other vaccine, we will have 74% power to find a significantdifference. If the true mortality rate is 10% for both monovalent andquadrivalent vaccines, with 15 mice/group we will have 69% power to shownon-inferiority of the quadrivalent formulation using a non-inferioritymargin of 30% (absolute difference), based on a 2-sided 95% confidenceinterval calculated by a likelihood score method.

Expected result—: It is likely that at least a single monovalentconjugate will be identified that induces high IgG titers withfunctional anti-bacterial properties by both the flagellin carrierprotein and OPS hapten, and will protect against wound infections withhomologous antigen expressing KP and PA pathogens. We anticipate that 3doses may be required to attain significant anti-OPS IgG levels. It is apossibility as well that equivalent immunogenicity and protection willbe seen between several OPS specific conjugate types in our monovalentpanel. If this occurs, our basis for down-selection will be forregulatory and manufacturing considerations. We also expect that aquadrivalent mixture will recapitulate the humoral responses seen formonovalent conjugates alone. Protection is presumed to be mediated byantibodies. Hence we further anticipate that passive transferimmunization with polyclonal KP-OPS:PA-Fla vaccine elicited sera willprotect against KP and PA.

Measuring protection in wound models in pigs using optimized conjugatevaccine formulations—The integumentary system of pigs is understood asthe best approximate of human skin, exhibiting similar architecture andstructural properties (Sullivan T P et al., Wound Repair Regen. 2001;9(2):66-76). Accordingly, the quadrivalent vaccine formulation developedin mice, is tested in a porcine full-thickness wound model.

The 50% effective dose (ED₅₀) for various doses of KP B5055 O1:1(2 andPA PAK are determined by infecting at various doses at multiple woundsites in naive pigs. Once a reliable infectious dose is determined, 4pigs 3 times are immunized with PBS or quadrivalent conjugate containing25 μg of total polysaccharide, as this approximate COPS dose was usedsuccessfully in human clinical trials for Shigella (Passwell J H, InfectImmun. 2001; 69(3):1351-1357; Cohen D, Infect Immun. 1996;64(10):4074-4077), S. Paratyphi A (Konadu E Y, Infect Immun. 2000;68(3):1529-1534), and E. coli (Ahmed A et al., J Infect Dis. 2006;193(4):515-521) COPS conjugates. As controls, 2 pigs are mock immunizedwith PBS alone. Twenty-one days after the final dose, immunized (2/individual pathogen) or control pigs (1/individual pathogen) areinfected at multiple sites with moderate or high levels of KP B5055 ofPA PAK (FIG. 34). Wounds sites will be isolated from each other, and canbe considered as independent.

Immunization and serological measurements—Thirty to 35 kg femaleYorkshire pigs are immunized intramuscularly on 3 occasions at 28 dayintervals as indicated. Sera is obtained before immunization and 21 daysafter the last dose. Anti-flagellin and anti-OPS serum IgG titers areassessed by ELISA, and functional antibodies are measured by OPA andmotility inhibition assays as described for Aim 2.

Porcine model of KP and PA wound infection—. A full dermal punch biopsyis used to generate a full thickness wound (beyond 0.7 mm) that passescompletely through the first layer of fat cells on the pig. Each animalreceives up to 48 wounds (3 groups of 16) using a 12 mm biopsy punch,along the back in the lumbar and thoracic area with each wound separatedby approximately 15 mm of normal skin. Animals are inoculated with ahigh or low dose of KP or PA and within 10 minutes of inoculation, allwounds will be covered with a dressing. At 1, 4, and 10 dayspost-infection, the dressings are removed, and 6 wounds per animal willbe analyzed for culture or biopsy. For each wound, two types of biopsiesare performed. For CFU/g tissue, a 6 mm punch biopsy are obtained. Forpathology, a sterile scalpel is used to obtain a full thickness wedgebiopsy. Additional biopsies may be taken based on previous cultureresults or wound appearance as appropriate. A similar biopsy on eachcollection day will be saved for scanning electron microscopy (SEM)evaluation of biofilm. Endpoint parameters will include wound size,CFU/g, clinical scores, biofilm formation, and histopathology toevaluate wound bed healing and re-epithelialization. Statisticalpower—Each wound is an independent observation. With 2 immunized animalsand 1 control animal for either KP or PA, we will have 6 and 3 totalindependent wounds, respectively, by which to measure pathology orburden for a particular dose of KP or PA. We will have 86% power to findstatistical significance, if the difference between the CFU/g means forimmunized relative to unimmunized pigs is 2.5 times the standarddeviation, which is assumed to be the same for both groups of pigs(2-sample t-test, α=0.05, 2-sided).

Expected results—: We anticipate that 3 doses of quadrivalent conjugatein pigs will induce 100% seroconversion for all vaccine components.Whereas our endpoint in mouse experiments is protection from mortality,the endpoint in pigs will be wound healing. Bacteremia and ascendinginfections are the major complication of PA and KP wound infections, andprotection against systemic spread is the primary target of our vaccine.Nevertheless, we anticipate that antibodies towards KP OPS and PA Flacould reduce overall tissue CFU/g through enhanced OPA by fixed tissuemacrophages and interference with biofilm formation. Hence, faster woundrecovery, improved tissue pathology and lower bacterial burden areexpected to be found.

While there have been shown and described what are presently believed tobe the preferred embodiments of the present invention, those skilled inthe art will realize that other and further embodiments can be madewithout departing from the spirit and scope of the invention describedin this application, and this application includes all suchmodifications that are within the intended scope of the claims set forthherein. All patents and publications mentioned and/or cited herein areincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated as having beenincorporated by reference in its entirety.

1. A conjugate comprising a Klebsiella surface polysaccharide antigenand a Pseudomonas flagellin protein or antigenic fragment or derivativethereof.
 2. The conjugate of claim 1, wherein the surface polysaccharideantigen comprises a polysaccharide antigen selected from the groupconsisting of an O polysaccharide (OPS), a core oligosaccharide and an Opolysaccharide (COPS), a capsule polysaccharide, and combinationsthereof.
 3. The conjugate of claim 1, wherein the surface polysaccharideantigen and the flagellin are covalently linked.
 4. The conjugate ofclaim 1, wherein the surface polysaccharide antigen is an Opolysaccharide antigen (OPS).
 5. The conjugate of claim 1, wherein thePseudomonas flagellin is selected from the group consisting of aPseudomonas aeruginosa flagellin type A (FlaA) and a Pseudomasaeruginosa type B (FlaB).
 6. The conjugate of claim 2, wherein theKlebsiella OPS is from a Klebsiella pneumoniae serovar selected from thegroup consisting of Klebsiella pneumoniae serovars O1, O2a, O3 and O5.7. The conjugate of claim 1, wherein the conjugate comprises i)Pseudomas aeruginosa flagellin type A (FlaA) or an antigenic fragment orderivative thereof and/or Pseudomas aeruginosa flagellin type B (FlaB)or an antigenic fragment or derivative thereof and ii) OPS fromKlebsiella pneumoniae selected from the group consisting of Klebsiellapneumoniae serovars O1, O2a, O3, O5 and combinations thereof.
 8. Theconjugate of claim 7, wherein Pseudomonas flagellin is covalently linkedto one or more OPS from a single Klebsiella pneumoniae serovar type. 9.The conjugate of claim 7, wherein the Pseudomas aeruginosa flagellintype A (FlaA) comprises SEQ ID NO:
 1. 10. The conjugate of claim 7,wherein the Pseudomas aeruginosa flagellin type B (FlaB) comprises SEQID NO:2.
 11. A vaccine composition comprising one or more conjugates ofclaim
 1. 12. The composition of claim 11, wherein the composition is amultivalent conjugate vaccine comprising one or more Pseudomonasflagellins covalently linked to one or more Klebsiella O polysaccharides(OPS).
 13. The composition of claim 11, wherein the conjugate vaccine isa multivalent conjugate vaccine comprising two different Pseudomonasflagellin or an antigenic fragment or derivative thereof covalentlylinked to one or more Klebsiella O polysaccharides (OPS).
 14. Thecomposition of claim 11, wherein the multivalent conjugate vaccinecomprises four different OPS antigens from Klebsiella pneumoniaeserovars O1, O2ab, O3, and O5.
 15. The composition of claim 11, whereinthe Pseudomonas is Pseudomonas aeruginosa.
 16. A method of inducing animmune response, comprising administering to a subject in need thereofan immunologically-effective amount of a conjugate comprising aKlebsiella surface polysaccharide antigen and a Pseudomonas flagellinprotein or an antigenic fragment or a derivative thereof.
 17. The methodof claim 16, wherein the surface polysaccharide antigen comprises apolysaccharide antigen selected from the group consisting of an Opolysaccharide (OPS), a core oligosaccharide and an O polysaccharide(COPS), a capsule polysaccharide, and combinations thereof.
 18. Themethod of claim 16, wherein the surface polysaccharide antigen and theflagellin are covalently linked.
 19. The method of claim 16, wherein thesurface polysaccharide antigen is an O polysaccharide antigen (OPS). 20.The method of claim 16, wherein multiple conjugates are administeredcomprising one or more Pseudomonas flagellins or antigenic fragments orderivatives thereof covalently linked to one or more Klebsiella Opolysaccharides (OPS).
 21. The method of claim 16, wherein the multipleconjugates comprise two different Pseudomonas flagellin covalentlylinked to one or more Klebsiella O polysaccharides (OPS).
 22. The methodof claim 21, wherein the two different Pseudomonas flagellins are aPseudomas aeruginosa flagellin type A (FlaA) and a Pseudomonasaeruginosa flagellin type B (FlaB).
 23. The method of claim 21, whereinthe multiple conjugates comprise four different OPS antigens fromKlebsiella pneumoniae.
 24. The method of claim 23, wherein the four OPSare from Klebsiella pneumoniae serovars O1, O2a, O3 and O5.
 25. Themethod of claim 21, wherein the two different Pseudomonas flagellins arePseudomas aeruginosa flagellin type A (FlaA) and Pseudomonas aeruginosaflagellin type B (FlaB) and the four Klebsiella OPS are from Klebsiellapneumoniae serovars O1, O2a, O3 and O5.
 26. The method of claim 25,wherein Pseudomonas flagellin is covalently linked to one or more OPSfrom a single Klebsiella pneumoniae serovar type.
 27. The method ofclaim 25, wherein the Pseudomas aeruginosa flagellin type A (FlaA)comprises SEQ ID NO:1.
 28. The method of claim 25, wherein the Pseudomasaeruginosa flagellin type B (FlaB) comprises SEQ ID NO:2.
 29. A methodof making the conjugate of claim 1, comprising covalently bonding aKlebsiella surface polysaccharide antigen and a Pseudomonas flagellinprotein or an antigenic fragment or a derivative thereof.
 30. The methodof claim 29, wherein the surface polysaccharide antigen is an Opolysaccharide (OPS).
 31. The method of claim 30, comprising covalentlybonding Pseudomonas aeruginosa flagellin type A (FlaA) and/or Pseudomasaeruginosa flagellin serotype B (FlaB) to at least one OPS fromKlebsiella pneumoniae serovars O1, O2a, O3 and O5.
 32. The method ofclaim 29, wherein the surface polysaccharide antigen is isolated from aKlebsiella pneumoniae serovar having one or more mutations.
 33. Themethod of claim 32, wherein the Klebsiella pneumoniae has an attenuatingmutation in the guaBA locus and a mutation in the wza-wzc locus. 34.(canceled)
 35. (canceled)
 36. (canceled)
 37. A Klebsiella pneumoniaeserovar strain, comprising an attenuating mutation in the guaBA locusand a mutation in the wza-wzc locus.
 38. The strain of claim 37, whereinthe serovar is selected from the group consisting of O1, O2a, O3, andO5.